Heat Shock-Induced Apoptosis in Preimplantation Bovine Embryos: MATERIALS AND METHODS(1)

Materials

Essentially fatty-acid free (EFAF) BSA was purchased from Sigma Chemical Company (St. Louis, MO). Bovine steer serum was from Pel-Freez (Rogers, AR). Modified Tyrode solutions were obtained from Cell and Molecular Technologies (Lavallette, NJ) to prepare Hepes-Tyrode albumin lactate pyruvate (TALP), in vitro fertilization (IVF)-TALP and Sp-TALP. Pituitary-derived FSH (Folltropin-V) was purchased from Ve-trepharm Canada Inc. (London, ON, Canada). Percoll was from Amersham Pharmacia Biotech (Uppsala, Sweden). Frozen semen from various bulls was purchased from American Breeders Service (Madison, WI) or was donated by Select Sires Inc. (Rocky Mount, VA). Potassium simplex optimized medium (KSOM) was obtained from Cell and Molecular Technologies.

The KSOM, which contains 1 mg/ml BSA, was modified on the day of use by adding an additional 2 mg/ml EFAF-BSA, 2.5 |xg/ml gen-tamicin, essential amino acids (basal medium Eagle), and nonessential amino acids (minimum essential medium) purchased from Sigma. Oocyte collection medium was tissue culture medium 199 (TCM-199) with Hanks salts without phenol red and supplemented with 2% (v/v) bovine steer serum (containing 2 U/ml heparin), 100 U/ml penicillin-G, 0.1 mg/ml streptomycin, and 1 mM glutamine. Oocyte maturation medium was TCM-199 with Earle salts supplemented with 10% (v/v) steer serum, 22 |xg/ml sodium pyruvate, 20 ^g/ml FSH, 2 |xg/ml estradiol-17p, 50 |xg/ml gen-tamicin, and an additional 1 mM glutamine.

The in situ cell death detection kit (fluorescein) and propidium iodide were obtained from Roche Diagnostics Corporation (Indianapolis, IN) and Sigma, respectively. Polyvinyl pyrrolidine (PVP) was purchased from Eastman Kodak Company (Rochester, NY). The Prolong Antifade Kit was obtained from Molecular Probes (Eugene, OR), RQ1 RNA-free DNase was from Promega (Madison, WI), and RNase A was from Qiagen (Valencia, CA). PhiPhiLux-G1D2 was obtained from OncoImmunin, Inc. (Gaithersburg, MD). Other reagents were purchased from Fisher (Pittsburgh, PA) or Sigma.

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