4 well dish

The methodology used for the in vitro maturation (IVM) of the bovine oocytes has been described elsewhere [20 (link)]. Cow ovaries were collected from a local slaughterhouse, placed in a thermos containing saline (0.9% w/v NaCl), and transferred to the laboratory at 35–37 °C within 1 h. The cumulus–oocyte complexes (COCs) were collected by aspiration of 3- to 8-mm follicles. Only those COCs with a compact cumulus and homogeneous dark ooplasm were selected for in vitro maturation (IVM). After adding 500 μL of maturation medium to each well in a 4-well dish (Nunc, Roskilde, Denmark), groups of up to 50 COCs were transferred to each well and cultured for 24 h. The composition of the maturation medium (IVM medium) was as follows: 10% (v/v) fetal bovine serum (FBS), 10 ng/mL epidermal growth factor and 50 μg/mL gentamicin added to tissue culture medium (TCM-199). All IVM procedures were performed in a 38.5 °C humid incubator with 5% CO₂.

IVM medium was prepared based on TCM-199 medium with Earle’s salts. It was buffered with 5.87 mmol/L HEPES and 33.09 mmol/L sodium bicarbonate, and supplemented with 0.1 g/L L-glutamine, 2.27 mmol/L sodium pyruvate, calcium lactate pentahydrate (1.62 mmol/L Ca²⁺, 3.9 mmol/L Lactate), 50 μg/mL gentamicin, 20% (v/v) fetal calf serum (FCS), 10 μg/mL of porcine follicle stimulating hormone (FSH), luteinizing hormone (LH; Pluset^®, Calier, Balcellona, Spain) [58 (link)], and 1μg/mL 17β estradiol [57 (link)]. IVM medium was pre-equilibrated for 1 h under 5% CO₂ in air at 38.5 °C, then loaded (400 µL/well) in a 4-well dish (Nunc Intermed, Roskilde, Denmark) and covered with pre-equilibrated lightweight paraffin oil. In each experiment, 20–25 COCs/well were added to a 4-well dish and cultured under conventional static IVM as control (CTRL) or loaded in a chamber of a commercial Live Box 1 (LB1) bioreactor (IVTech S.r.l.—Massarosa, Italy) and used to test different mIVM systems, as described in Table 1. In each experiment, in vitro culture was performed by placing the bioreactor, together with the control 4-well plate, in the incubator for 24 h at 38.5 °C under 5% CO₂ in air.

We followed the guidelines of Animal Research Institute Committee of Nanjing Agricultural University to conduct the operations. The animal facility had license authorized by the experimental animal committee of Jiangsu Province (SYXK-Su-20170007). The Female Institute of Cancer Research (ICR) mice, aged 4–6 weeks, were kept in a room with a regulated temperature of 22°C and provided with a standard diet. Fully developed GV stage oocytes were retrieved from the ovaries of mice, and then cultured in M16 medium with paraffin oil at 37°C and in the presence of 5% CO₂ for in vitro maturation. At specific intervals, the oocytes were collected for various tests and analyses. The oocytes were placed at 37°C with an atmosphere of 5% CO₂, and cultured to different time points for immunostaining, microinjection, and western blot.
For porcine oocyte collection, the porcine ovaries were delivered from a local slaughterhouse in the thermos bottle within 2 hr. The cumulus cell complex (COCs) were acquired from 3 to 6 mm antral follicles, and cultured in TCM-199 medium for in vitro maturation from 4-well dish (Nunc, Denmark) at 38.5°C with an atmosphere of 5% CO₂. The porcine oocytes were collected at 24–28 hr for MI stage and 44–48 hr for MII stage.

Porcine ovaries obtained from a slaughterhouse were delivered to the laboratory in a thermos containing 0.9% saline and at 36 °C. Within 2 h, 3–6-mm ovarian follicles were aspirated using a 10-mL syringe with an 18-gauge needle. The obtained porcine follicular fluid was centrifuged at 500×g for 30 min at 4 °C, and then filtered through 0.45-μm filters. Cumulus-oocyte complexes (COCs) were washed using TLH-PVA medium [HEPES-buffered Tyrode’s medium (TLH) containing 0.05% (w/v) polyvinyl alcohol (PVA)], and then co-cultured with 50–60 COCs per well in a 4-well dish (Nunc, Roskilde, Denmark) containing 500 μL of IVM medium. IVM was performed in IVM medium containing 10 IU/mL equine chorionic gonadotropin and 10 IU/mL human chorionic gonadotropin for 22 h, and then the cells were moved to hormone-free IVM medium and cultured for 18 h. The composition of IVM medium was TCM199 (Gibco), 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng/mL epidermal growth factor, 75 μg/mL kanamycin, 1 μg/mL insulin, and 10% (v/v) porcine follicular fluid. The incubation conditions for IVM were 39 °C and 5% CO₂ in a humid incubator (Astec). Mature COCs were denuded by gently pipetting with 0.1% hyaluronidase, and then washed with TLH-PVA medium. MII oocytes obtained by this process were used for subsequent experiments.