A1603

Sodium alginate (2.5 g) and 50 ml distilled water were mixed in a Sakaguchi flask (500 ml) at 120 rpm and 37 °C for 16 h to obtain a uniformly dispersed, clear alginate gel containing 50 g/l sodium alginate. We added 40 ml of 0.85% NaCl and 1 mg/l commercial alginate lyase (A1603; Sigma-Aldrich, St. Louis, MO, USA) with shaking at 120 rpm and 37 °C for 25 h. Depolymerized alginate solution was sterilized using Nalgene Rapid-flow filters (pore size 0.2 μm; Thermo Fisher Scientific, Waltham, MA, USA).

On the 6th day of culturing, follicles were released from alginate beads using alginate lyase (A1603, Sigma-Aldrich) and incubated in maturation media (αMEM with 10% FBS, 1%PS, 1.5 IU/ml human chorionic gonadotropin, 10 ng/ml epidermal growth factor [EGF] [PHG0311, Gibco]) for 18 h. After 18 h of incubation, ruptured follicles, ovulated COCs and oocytes were imaged, and the ovulation rate was calculated by observing the ovulation of 7 follicles. The follicular wall of ovulated follicles was ruptured, with ovulated cumulus–oocyte-complex (COC) around. Oocytes with first polar body extrusion were classified as mature oocytes. Every 3 follicles were collected for RNA extraction using RNeasy Mini Kit (74104, QIAGEN).