The strength of the protein signal is referred to as relative expression because the total amount of protein analyzed was similar for MG132 and vehicle-treated oocytes. Differentially expressed proteins were analyzed for GO terms by Blast2GO [14] and, after conversion to official gene symbols, by the Database for Annotation, Visualization and Integrated Discovery [DAVID; (DAVID Bioinformatics Resources 6.7, http://david.abcc.ncifcrf.gov/)] [15]. For DAVID, genes were annotated using the bovine genome as a reference. In addition, functional properties of differentially-abundant proteins were determined by mining PUBMED using the Chilibot program (www.chilibot.net) [16].an epidural block (5 mL 2% lidocaine, w/v) before transfer. Pregnancy was diagnosed by ultrasound at d 32 and by rectal palpation at d 46 and 71. A total of 24 embryos produced with vehicle and 30 embryos produced with MG132 were transferred. Data on cleavage and blastocyst development were analyzed by least-squares analysis of variance as described for Experiments 1? (n = 10 replicates) while data on pregnancy rate were analyzed by chi-square analysis.
Results Concentration-dependent Effect of MG132 from 16?2 h of Maturation on Subsequent Embryonic Development (Experiments 1 and 2)
In the first experiment, COCs were treated from 16 to 22 h of maturation with 0, 1, 5 or 10 mM MG132 (Table 1). The highest concentration of MG132 increased (P,0.05) the percentage of oocytes that cleaved (i.e., that were $2 cells) and the percentage of oocytes that became blastocysts. There was, however, no effect of 10 mM MG132 on the percentage of cleaved embryos that became blastocysts or on the number of cells per blastocyst. There were also no effects of lower concentrations of MG132 on any endpoint. In Experiment 2, COCs were treated with 0, 10, 20 or 30 mM MG132 (Table 2). As shown in Experiment 1, treatment of COCs with 10 mM MG132 increased cleavage rate and the percentage of oocytes becoming blastocysts (P,0.05). In addition, the percentage of cleaved embryos becoming blastocysts was increased (P,0.05) by treatment with 10 mM MG132. As in Experiment 1, there was no effect of 10 mM MG132 on blastocyst cell number. Treatment with 20 mM MG132 increased (P,0.05) cleavage rate but did not affect other endpoints examined. Treatment with 30 mM MG132 had no effect on subsequent development.
Pregnancy Rates after Transfer of Embryos Produced using MG132 During Oocyte Maturation (Experiment 8)
Embryos were produced in vitro using Holstein COCs that were collected from abattoir-derived ovaries (Central Packing, Center Hill, FL). Maturation was carried out using conditions similar to those for other experiments. At 16 h of maturation, COCs were washed and then placed in fresh medium containing 10 mM MG132 or vehicle. Fertilization was carried out for 8 h in SOF-IVF [17] using X-sorted semen from a single Holstein bull (Accelerated Genetics, Baraboo, WI, USA and Select Sires, Plain City, OH, USA). A total of 4 different bulls were used in the experiment. Sperm were purified before fertilization as described elsewhere [18]. The final sperm concentration in the fertilization well was 16106 sperm/ml. Following fertilization, presumptive zygotes were cultured in 50 ml microdrops of BBH7 culture medium overlaid with mineral oil in groups of 25?0 in a humidified atmosphere of 5% CO2 and 5% O2 (balance N2) at 38.5uC. Grade 1 expanded blastocysts [19] were harvested at d 7 after insemination and vitrified using the open-pulled straw method as described elsewhere [18]. On the day of transfer, open-pulled straws were thawed and contents emptied into a 2-well plate (Agtech, Manhattan, KS, USA) filled with thaw medium [Tissue Culture Medium 199 with Hank’s salts and supplemented with 10% (v/v) fetal bovine serum and 50 mg/mL gentamicin] containing 0.33 M sucrose. Immediately afterwards, embryos were transferred to a fresh well of the same medium. Embryos were then loaded individually into 0.25 mL embryo transfer straws and transferred immediately thereafter (,5 min after thawing). Embryos were transferred to lactating female Holsteins on four occasions between June 10, 2011 and August 19, 2011 at the University of Florida Dairy Unit (Hague, FL; 29.77904 N, 82.48001 W). Cows were housed in free-stall barns equipped with fans and sprinklers. They were fed a total mixed ration and milked two times per day. Cows were either first-service cows or cows that had previously been inseminated or received an embryo during that lactation and had been diagnosed non-pregnant. Cows were subjected to the Ovsynch-56 timed ovulation protocol [20]. Specifically, cows received 100 mg gonadotropin releasing hormone (GnRH), i.m., on d ?0; 25 mg prostaglandin F2a (PGF), i.m., on d ?; and 100 mg GnRH, i.m., at 56 h after PGF. For first-service cows only, the timed ovulation protocol was preceded by a presynchronization protocol (two injections of 25 mg PGF, i.m., 14 d apart), with the last injection 12 d before initiation of the Ovsynch-56 protocol. Embryos were transferred on day 7 of the above-mentioned synchronization protocol to cows diagnosed by ultrasonography as having a corpus luteum on the scheduled day for embryo transfer. Each cow received a single embryo in the uterine horn ipsilateral to the corpus luteum. Cows were paired and randomly assigned within pair to receive an embryo produced with vehicle or MG132.
Actions of MG132 During the First Six or Last Six Hours of Maturation (Experiment 3)
Results of Experiments 1 and 2 indicated that the response of COCs to MG132 occurred over a narrow range and that optimal effects on maturation were achieved with 10 mM MG132. Consequently, subsequent experiments were performed with MG132 at this concentration. For Experiment 3, it was tested whether MG132 would affect maturation differently when added at 0? h of maturation, when proteasomes are necessary for completion of meiosis I, than when added at 16?2 h of maturation (Table 3). When added from 0? h, addition of MG132 reduced the proportion of oocytes that cleaved and the proportion of oocytes and cleaved embryos that became blastocysts (main effect of MG132, P,0.01 or less). Addition of MG132 from 16?2 of maturation increased (P,0.05) the percentage of oocytes undergoing cleavage. MG132 from 16?2 h also increased the percentage of oocytes and cleaved embryos developing to the blastocyst stage provided COCs were not also treated with MG132 from 0? h (interaction, P,0.05). Addition of MG132 from 16?2 h increased (P,0.02) blastocyst cell number slightly but differences were not detected using the pdiff mean separation test.
