Female Infertility Clinic Protocols
In vitro Maturation
Ovaries are manually macerated with 25 gage needles and oocyte/cumulus cell complexes are collected with a finely drawn micropipette. Complexes are washed in fresh minimum essential medium (MEM) prepared with Earle’s balanced salt solution, both essential and nonessential amino acids (Gibco, Grand Island, NY, 0.23 mM pyruvic acid, 75 mg/L penicillin G, 50 mg/L streptomycin sulfate, 0.01 mM tetra sodium EDTA (Sigma), 3mg/ml BSA (Sigma), and 5% Fetal Bovine Serum (Sigma). The vitamins and the essential and non-essential amino acids were added from 100-strength or 50-strength concentrates. Complexes are incubated and allowed to mature 16-17 hours in 2.5 ml of medium. Incubation takes place at 37C in a modular incubation chamber (Billups Rothenberg, Del Mar, CA) thoroughly infused with a gas mixture composed of 5% O2:5% CO2:90% N2. After maturation, cumulus cells are removed from the oocytes by drawing complexes in and out of a Pasteur pipette. The oocytes that have undergone germinal vesicle break down are rinsed 3X and fertilized in vitro.
In vitro Fertilization and Development
Mature oocytes are fertilized in vitro using epididymal sperm from 3-6 month old males that have been housed individually since weaning. Fertilization is carried out for 4 hours in MEM without Fetal Bovine Serum (see above for media and incubation conditions). After incubation for 4 hours, the oocytes are washed twice in fresh medium and then cultured overnight in 1 ml of the same medium. Embryos that cleave to the 2-cell stage are collected and washed twice in KSOM supplemented with 0.5-strength essential and nonessential MEM amino acids; they are then cultured until the blastocyst stage in this medium.
References:
Eppig JJ, O’Brien MJ. Development in vitro of mouse oocytes from primordial follicles. Biology of Reproduction (1996) 54: 197-207
O’Brien MJ, Pendola JK, Eppig JJ. A Revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biology of Reproduction (2003) 68:1682-1686.
Eppig JJ, Wigglesworth K Atypical maturation of oocytes of strain I/LnJ mice. Molecular Reproduction and Development (1994) 9:1136-1142.
Ho Y, Wigglesworth K, Eppig JJ, Schultz RM. Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Molecular Reproduction and Development (1995) 41:232-238.
