Medium 199

In vitro production of bovine embryos, including in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC), was performed as published before (Cui et al. 2016 (link), Li et al. 2021 (link), Shi et al. 2021 (link)). Briefly, cumulus–oocyte complexes (COCs) containing intact cumulus cells were collected from bovine ovaries obtained from a local abattoir and these COCs were matured in the IVM medium at 38.5°C under 5% CO₂ in humidified air for 22–24 h. The IVM medium consists of 10% fetal bovine serum (FBS; Gibco-BRL), 1 IU/mL follicle-stimulating hormone (Sansheng Biological Technology, Ningbo, China), 0.1 IU/mL luteinizing hormone (Solarbio, Beijing, China), 1 mM sodium pyruvate (Thermo Fisher Scientific), 2.5 mM GlutaMAX™ (Thermo Fisher Scientific), and 10 μg/mL gentamicin in Medium-199 (M4530). After maturation, COCs (60–100 COCs per well in 4-well plates) were then incubated with spermatozoa (1 × 10⁶–5 × 10⁶) purified from frozen-thawed semen using a percoll gradient in BO-IVF medium (IVF Bioscience, Falmouth, Cornwall, UK). The IVF condition was 38.5°C under 5% CO₂ for 9–12 h. Putative zygotes were then removed from cumulus cells by pipetting up and down in Medium-199 (M7528) supplemented with 2% FBS (Gibco-BRL). Embryos were incubated in BO-IVC medium (IVF Bioscience) at 38.5°C under 5% CO₂ in humidified air until use.

To culture epithelial cells, six 5-months old lenses from CRYαA_WT - and CRYαA_N101D mice were excised and incubated with 0.25% trypsin at 37^ο C for 2.5 h in an incubator with 5% CO₂-humidified air. Next, the lens cells in trypsin solution were centrifuged at 1200 rpm for 3 min, and trypsin (in the supernatant) was discarded. The lens epithelial cells (recovered as pellet) were suspended in medium 199 (Thermo Fisher Scientific, Grand Island, NY) containing 10% fetal calf serum (Hyclone, Logan, Utah) and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific, Grand Island, NY) in 12-well plates (Corning, Franklin Lakes, NJ). After 24 h, the unattached cells were discarded by washing with the above medium. The old medium was replaced with fresh medium after every 48 h, and the cells were allowed to grow for 7 to 10 days until confluent. Next, the confluent cells were trypsinized and seeded in 12-well plates for intercellular Ca²⁺ determination and were allowed to grow for 24 h. The cells were washed with medium 199 without phenol red, incubated in calcium orange dye (Thermo Fisher Scientific, Grand Island, NY) at a final concentration of 4 μM for 30 min at room temperature as instructed in the manufacturer’s protocol. After 30 min, the cells were washed with the above medium, and Ca²⁺ indicator was examined under a microscope (Leica DMI 4000B) using a Texas Red filter.