Culture Media
Chemicals were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) or Fisher (Pittsburgh, PA, USA) unless otherwise stated. The base medium for oocyte maturation (OMM) was Tissue Culture Medium-199 (TCM-199; Invitrogen, Carlsbad, CA) with Earle’s salts supplemented with 10% (v/v) bovine steer serum containing 2 U/ml heparin (Pel-Freez, Rogers, AR, USA), 2 mg/ml estradiol 17-b, 20 mg/ml bovine follicle stimulating hormone (Folltropin-V; Bioniche Life Sciences, London, ON, Canada), 22 mg/ml sodium citrate, 50 mg/ml gentamicin sulfate and 1 mM glutamine. Oocyte collection medium (OCM) was TCM-199 medium with Hank’s salts (Cellgro, Mediatech, Manassas, VA, USA) supplemented with100 U/ml penicillin-G, 0.1 mg/ml streptomycin, 1 mM glutamine and 2% (v/v) bovine steer serum containing 2 U/ml heparin. HEPES-Tyrodes albumin lactate pyruvate solution (TALP) was prepared as described previously [8]. The fertilization medium was in vitro fertilization (IVF)-TALP [8]. Percoll was from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Frozen semen from bulls of various breeds was donated by Southeastern Semen Services (Wellborn, FL, USA). The embryo culture medium was SOF-BE1 [9] or, for one experiment, a proprietary culture medium called BBH7 from Minitube (Verona, WI, USA). Hoechst 33342 was purchased from Sigma-Aldrich. The MG132 was purchased from Sigma-Aldrich.
dish (Minitube, Verona, WI, USA). Oocytes were fertilized with a pool of frozen-thawed sperm from three bulls purified by Percoll gradient centrifugation [8]; different pools were used in each replicate. Oocytes were fertilized (day of fertilization termed Day 0) by adding 30 ml of Percoll-purified spermatozoa (final concentration in fertilization dish = 16106 sperm cells/ml in IVF-TALP and 20 ml PHE (0.5 mM penicillamine, 0.25 mM hypotaurine and 25 mM epinephrine in 0.9% (w/v) NaCl). After 8 h at 38.5uC and 5% (v/v) CO2 in humidified air, putative zygotes were removed from fertilization wells, denuded of cumulus cells by vortexing in hyaluronidase (10,000 U/ml in 600 ml HEPES-TALP) for 4 min, washed in HEPES-TALP, and placed in groups of 20 to 35 putative zygotes in 50 ml microdrops of SOF-BE1 medium overlaid with mineral oil at 38.5uC in a humidified atmosphere of 5% (v/v) CO2, 5% O2 and the balance nitrogen. Cleavage and blastocyst formation were evaluated on Days 3 and 8 after IVF, respectively.
Examination of Nuclear Status of Oocytes after IVM
At 16 h and 22 h of IVM, COCs were transferred into HEPESTALP containing 0.3% (w/v) hyaluronidase and then vortexed for 5 min to remove cumulus cells. Denuded oocytes were stained with 5 mg/ml Hoechst 33342 in 10 mM PBS (10 mM PO4, 0.9% (w/v) NaCl) containing 1% (w/v) polyvinylpyrrolidone (PBS-PVP) for 1 h at room temperature. Then, 10?5 oocytes were mounted on glass slides with a small amount of anti-fade solution (Life Technologies, Grand Island, NY, USA and covered with a cover slip. Oocytes were examined using an Axioplan 2 epifluorescence microscope (Zeiss, Gottingen, Germany) with blue filter (excitation ?wavelength = 365/12 nm; emission wavelengths = 395?50 nm). Each oocyte was classified according to stage of nuclear maturation as germinal vesicle (GV), germinal vesicle break down (GVBD), pre-metaphase I-metaphase I (MI), anaphase I (AI)telophase I (TI) and metaphase II (MII).
Examination of Pronuclear Status of Oocytes after IVF
Inseminated oocytes were harvested from culture drops of SOFBE1 at 18 h after fertilization, mounted on glass slides and nuclei visualized using Hoechst 33342 as described above for oocytes. Oocytes were classified as non-penetrated if the nucleus was at MI or MII without the presence of a sperm head or male pronucleus. An oocyte was classified as fertilized if one swollen sperm head or male pronucleus was detected inside the oocyte. Oocytes having more than one swollen sperm head or male pronuclei were classified as polyspermy.
Oocyte Collection and In Vitro Maturation (IVM)
Bovine ovaries were obtained from various breeds at a local abattoir (Central Packing, Center Hill, Florida) and transported to the laboratory. The owner provided permission to use the ovaries for experimental purposes. Cumulus-oocyte complexes (COCs) were collected by slicing superficial follicles (2?0 mm in diameter) with a scalpel blade and washing the ovaries into a beaker containing OCM. The COCs were harvested using a capillary pipette and washed three times in OCM. Groups of 10 were placed into 50 ml drops of OMM covered with mineral oil. The COCs were matured for 22 h at 38.5uC in a humidified atmosphere of 5% (v/v) CO2, with MG132 treatment applied according to the experimental design. The MG132 was dissolved in dimethyl sulfoxide (DMSO) and was added to maturation drops so that the final concentration of DMSO was not greater than 0.5% (v/v). The control oocytes were cultured with medium supplemented with a similar amount of DMSO during IVM as for oocytes treated with MG132.
Blastocyst Cell Number
Blastocysts were fixed for 1 h at room temperature in 4% (w/v) paraformaldehyde dissolved in PBS. After washing in PBS-PVP, embryos were incubated with 1 mg/ml Hoescht 33342 dissolved in PBS-PVP. Embryos were washed in PBS-PVP, placed on a microscope slide and number of nuclei counted using a Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Gottingen, Ger?many).
Experiments on Oocyte Maturation, Fertilization and Development (Experiments 1?)
The concentration-dependent effects of MG132 added at the end of oocyte maturation on embryonic development were tested in Experiments 1 and 2. COCs were matured in OMM that was supplemented with 0, 1, 5, 10 mM MG132 (Experiment 1) or 0, 10, 20 or 30 mM MG132 (Experiment 2) from 16 h to 22 h after initiation of maturation. Treatment was achieved by washing
In Vitro Fertilization and Culture
After maturation, all COCs were washed twice in HEPESTALP and once in IVF-TALP and then transferred in groups of 30?0 oocytes to 425 ml of fertilization medium in wells of a 5-wellCOCs after 16 h of maturation and placing them in fresh medium containing MG132 or vehicle. Endpoints were cleavage rate at day 3 after insemination, the proportion of oocytes and cleaved embryos that became blastocysts at Day 8, and blastocyst cell number. The experiments were replicated six times with 20?0 COCs per treatment for each replicate (Experiment 1) and four times with 20?0 COCs per treatment for each replicate (Experiment 2). Experiment 3 was conducted to determine whether timing of MG132 treatment altered effects of the inhibitor on embryonic development. COCs were untreated or treated with 10 mM MG132 at two times [0? h of maturation (during the initiation of maturation) or 16?2 h of maturation (at the end of maturation)] using a 262 factorial arrangement of treatments. The COCs were placed in appropriate treatments at 0 h (vehicle or MG132), washed at 6 h, placed in fresh medium without MG132, washed at 16 h of maturation, and placed in fresh medium with appropriate treatment. Thus, some cultures received vehicle at 0? h and 16?22 h, some received MG132 from 0? h and 16?2 h, some received MG132 from 0? h and vehicle from 16?2 h, and some received vehicle from 0? h and MG132 from 16?2 h. Endpoints were cleavage rate at day 3 after insemination and the proportion of oocytes and cleaved embryos that became blastocysts at Day 8. The experiment was replicated six times with 20?0 COCs per treatment for each replicate.
