Beneficial effects of MG132 on nuclear remodeling, transcript abundance and embryonic development have also been shown for embryos constructed by somatic cell nuclear cloning in mice [22,23], rats [24,25], goats [23] and pigs [7,26,27]. Unlike for addition from 0? h, MG132 added from 16?2 h did not improve oocyte competence by improving nuclear maturation because the percentage of oocytes that were MII at the end of maturation was not affected by MG132 later in maturation. Rather, some of the beneficial effect of MG132 from 16?2 h on the percentage of oocytes that Data are least-squares means 6 SEM of values from six replicates. Values in the same column with different superscript letters are significantly different (P,0.05). e N.S. = non-significant (P.0.10).

Table 5. Effect of treatment with 10 mM MG132 from 0? or 16?2 h of maturation on meiotic maturation at 22 h after initiation of maturation (Experiment 5).Data are least-squares means 6 SEM of values from three replicates. GVBD: germinal vesicle break down; MI: metaphase I; Ana-Telo: anaphase ?telophase; MII: metaphase II. c,d,e Values in the same column with different superscript letters are significantly different (P,0.05 or, for). f N.S. = non-significant (P.0.10).became blastocysts was due to 1) increased cleavage rate through actions not involving fertilization rate and 2) increased competence of the fertilized oocyte to develop to the blastocyst stage. Indeed, the potential of a newly formed embryo to become a blastocyst was improved by addition of MG132 from 16?2 h in two of three experiments evaluated, as indicated by a significant improvement in the percentage of cleaved embryos that became blastocysts. The mechanism by which MG132 late in maturation improves competence of the oocyte to support development is likely to involve arrest of processes mediated by proteasomes that ordinarily compromise the oocyte. One result is likely to be increased transcript abundance for genes required for embryonic development, as shown in the pig oocyte [7]. In the mouse, MG132 improved oocyte competence in aged oocytes but did not affect non-aged oocytes [6]. It might be that MG132 blocked proteasome-mediated degenerative changes in a portion of maturing oocytes of inferior quality caused by prolonged culture during maturation or other reasons. Proteomic analysis was performed to determine possible targets of proteasomal cleavage whose relative expression was altered by MG132 treatment from 16?2 h. Such proteins might be involved in the beneficial effects of MG132 on oocyte competence and may
be important molecules for determining the ability of an oocyte to complete the first cleavage division and support development of the embryo to the blastocyst stage. One limitation to the experimental approach was that less abundant proteins were less likely to be detected by mass spectrometry. Nonetheless, a total of 653 proteins could be analyzed for differences in amount between oocytes treated with vehicle or MG132. Surprisingly, there were a greater number of proteins whose relative expression was decreased by MG132 than there were proteins that were increased. Regulation of intracellular proteins in the presence of MG12 is complex. In HEK293T cells, MG132 can increase ubiquitination of some proteins and decrease ubiquitination of others [28]. Some proteins in the bovine oocyte increase in abundance during oocyte maturation whereas others decline in amount [29]. It is possible that inhibition of the proteasome by MG132 late in maturation protected some proteins from proteolysis, which in turn hastened or exaggerated the maturation-dependent decline in other oocyte proteins. Six of the proteins that were decreased by MG132 (ADSL, AHCY, CDK5, GSTM3, STIP1, and THOP1) and two that were increased by MG132 (CAND1 and GAPDH) are encoded for by transcripts that decrease during nuclear maturation of bovine oocytes [30].

Table 6. Effect of treatment with 10 mM MG132 from 0? or 16?2 h of maturation on fertilization rate (Experiment 6).a MG132, 0? h No No Yes Yes Probability of treatment effects MG132, 0? MG132, 16?2 Interaction Data are least-squares means 6 SEM of values from four replicates. N.S. = non-significant (P.0.10). Figure 1. Expression levels and detection of peptide of Cullin-associated NEDD8-dissociated protein1 (CAND1). Panel A: Mean 6 SEM of CAND1 expression for control and MG132-treated oocytes. There was a difference (P = 0.004) between treatments. Panel B: Reporter ion expression for the C peptide fragment of CAND1. 114 and 115 represent two separate biological replicates of control oocytes while 116 and 117 represent two separate biological replicates of MG132-treated oocytes. Panel C: b and y ions and amino acid sequence from one peptide fragment of CAND1. Among the oocyte proteins regulated by the proteasome are proteins involved in RNA processing [2,3] so inhibition of proteasomal activity with MG132 could affect stability and translation of a variety of mRNA. There were 6 annotated proteins identified whose relative expression was increased by MG132 (ACAT1, CAND1, TUBACA1C, P4HB, HYOU1, and GAPDH). The increase in GAPDH may be a direct result of inhibition of the proteasome because intracellular amounts of GAPDH are regulated by ubiquitination [31,32]. Another mechanism may be involved in regulation of CAND1 by MG132. This protein interferes with ubiquitin ligase activity [33]. Perhaps, inhibition of cleavage of ubiquitinated proteins leads to increased synthesis or decreased degradation of CAND1 through feedback mechanisms. Other proteins involved in the ubiquitin pathway were decreased by MG132, notably HSP90B1, THOP1, UBA1, and VCP. None of the 6 annotated proteins increased by MG132 have been identified as a marker of oocyte competence. Nonetheless, an increase in amounts of these proteins could potentially affect oocyte competence. GAPDH, for example, catalyzes an important step in glycolysis. Glycolysis in the bovine oocyte is low and most pyruvate for the oocyte is supplied by the surrounding cumulus cells [34]. There is some evidence, though, that rate of glycolysis in the bovine oocyte is proportional to developmental competence[35]. Another protein increased by MG132 was TUBA1C. Tubulins are important for organelle movement in the oocyte and completion of meiosis [36,37]. Two other upregulated proteins, P4HB and HYOU1, function in protein folding [38,39].