Experiments 4? were conducted to determine effects of MG132 on oocyte nuclear maturation (Experiments 4 and 5) and fertilization rate (Experiment 6). For Experiment 4, COCs were treated with vehicle or 10 mM MG132 for the first 6 h of maturation and nuclear maturation was examined at 16 h after initiation of maturation. The experiment was replicated three times with 20?5 COCs per treatment for each replicate. For Experiments 5 and 6, COCs were untreated or treated with 10 mM MG132 at two times (0? h of maturation, 16?2 h of maturation, or at both times) using a 262 factorial arrangement of treatments and procedures as described for Experiment 3. The endpoints were nuclear maturation at 22 h of maturation (Experiment 5) or sperm penetration at 18 h after exposure to sperm (Experiment 6). Experiment 5 was replicated three times with 20?5 COCs per treatment for each replicate. Experiment 6 was replicated four times with 20?0 COCs per treatment for each replicate. Data were analyzed statistically as follows. For each replicate, percentage data (for example, percentage of oocytes that cleaved and percentage of cleaved embryos that became blastocysts) were calculated for all oocytes or embryos within the same treatment. Thus, the group of oocytes treated alike within each replicate was the experimental unit. Statistical analyses were performed using the Statistical Analysis System (version 9.2; SAS Institute Inc., Cary, NC, USA). Data were analyzed using the General Linear Models procedure. For main effects with more than 1 degree of freedom, the pdiff mean separation procedure was used when main effects or interactions differed at P,0.10. Percentage data were arcsine-transformed prior to analysis to maintain homogeneity of variance. Results are expressed as least-squares means 6 standard error of the mean (SEM) of the untransformed data.

Effect of MG132 on the Oocyte Proteome (Experiment 7)
Oocytes were matured as described above. After 16 h of maturation, COCs were placed in fresh medium containing 10 mM MG132 or vehicle. The COCs were denuded after 22 h of maturation by vortexing after treatment with hyaluronidase. Those oocytes in which a polar body was evident by light microscopy were retained and processed for protein extraction.
The zona pellucida was removed by treatment for 5 min with 0.1% (w/v) protease from Streptomyces griseus followed by mechanical shearing. Three biological replicates were included for both vehicle and MG132 groups. A biological replicate represented a pool of polarbody-extruded oocytes collected from several oocyte maturation procedures. The number of oocytes per pool was 225 for replicate 1, 225 for replicate 2 and 1000 for replicate 3. Oocytes were suspended in 10 mM KPO4, pH 7.4 containing 1 mg/ml polyvinyl alcohol and 1% (w/v) protease inhibitor cocktail (Sigma) and stored at 270uC until processing. Total protein was isolated from pooled oocytes and purified as described elsewhere [10]. The protein concentration was determined using the BCAH Protein Assay (Thermo, Rockford, IL, USA). For each sample (regardless of the number of starting oocytes), 100 mg protein was dissolved in protein buffer [0.2% (w/v) sodium dodecyl sulfate, 8 M urea, and 10 mM Triton X-100). The samples were reduced, alkylated, trypsin-digested, and labeled following the manufacturer’s instructions for the iTRAQ Reagents 4-plex kit (AB Sciex Inc., Foster City, CA, USA). To verify the tag efficiency of the iTRAQ method, iTRAQ tags 114 and 115 were used to label control samples and tags 116 and 117 were used to label MG132 groups. Two iTRAQ procedures were conducted. In Set 1, one control and one MG132 sample were analyzed twice to determine technical replication. In Set 2, two biological replicates of each treatment were analyzed. Proteins were identified using an off-line 2D liquid chromatography-MS/MS method with strong cation exchange (SCX) chromatography as a first step to fractionate the oocyte proteome (Figure S1). The tryptic peptide mixtures were lyophilized, dissolved in SCX solvent A [25% (v/v) acetonitrile, 10 mM ammonium formate, and 0.1% (v/v) formic acid, pH 2.8], and fractionated using an Agilent HPLC system 1260 with a polysulfoethyl A column (2.1 6 100 mm, 5 mm, 300 ?A; PolyLC, Columbia, MD, USA). Tryptic peptides were separated with a LC Packing C18 Pep Map HPLC column (Dionex, San Francisco, CA, USA), and a hybrid quadrupoleTOF QSTAR Elite MS/MS system (AB Sciex Inc., Framingham, MA, USA) was used for data acquisition.

The MS/MS data were processed by a thorough search considering biological modification and amino acid substitution against the National Center for Biotechnology Information nonredundant Bos taurus fasta database (83,655 entries) and uniprot B. taurus database (33,808 entries) under the ParagonTM algorithm [11] using ProteinPilot v.4.2 software (Applied Biosystems). After searching MS/MS spectra against these databases, results were combined into each group. Animal species, fixed modification of methylmethane thiosulfate-labeled cysteine, fixed iTRAQ modification of amine groups in the N-terminus and lysine, and variable iTRAQ modifications of tyrosine were considered. The ProteinPilot cutoff score was set to 1.3 (a confidence level of 95%), and the false discovery rate (FDR) was estimated by performing the search against concatenated databases containing both forward and reverse sequences (Table S1). For protein quantification, we only considered MS/MS spectra that were unique to a particular protein and where the sum of the signal-to-noise ratio for all of the peak pairs was .9 (software default settings, AB Sciex Inc.). The accuracy of each protein ratio was calculated by the ProGroup analysis in the software to determine whether the protein is significantly differentially expressed [12]. To be identified as being significantly differentially expressed, a protein must have been quantified with at least three spectra, the fold change was .1.2 or ,0.8, and the P value for vehicle vs MG132 was ,0.05 as determined with Fisher’s combined probability test [13] (Fisher, 1948).