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The objective of this study was to assess the practical usefulness of morphologically poor oocytes (MPCOCs) in relation to follicular size and oocyte diameter. Oocytes collected from medium (3-8 mm in diameter) and small (<3 mm) follicles were classified into five categories of morphologically good oocytes (MGCOCs) from medium follicles (MA, control), MPCOCs with larger and smaller diameters from medium follicles (ML and MS, respectively), and those from small follicles (SL and SS, respectively). The oocytes were examined for maturation and developmental competence after parthenogenesis and somatic cell nuclear transfer (SCNT). Nuclear maturation of ML oocytes (91%) was similar to that of control oocytes (94%), but higher than MS (80%), SL (79%), and SS (63%) oocytes. This pattern was also observed in the intracellular glutathione level, p34(cdc2) kinase activity, and gene (CDK1, PCNA, and ERK2) expression levels in in vitro-matured oocytes. ML oocytes showed a similar proportion of blastocyst formation (20%) after SCNT to control oocytes (21%). In addition, the use of ML oocytes resulted in a 50% farrowing rate with 1.8% efficiency of piglet production after SCNT embryo transfer, while control oocytes showed a 60% farrowing rate with 2.4% production efficiency. Our results demonstrate that MPCOCs, if appropriately selected, have a comparable ability to MGCOCs in supporting not only in vitro blastocyst formation, but also development to term in vivo after SCNT. These oocytes can be used as a source for in vitro production of embryos with normal in vivo viability in pigs.

Maturation of the oocyte involves nuclear and cytoplasmic changes that include post-translational processing of proteins. The objective was to investigate whether inhibition of proteasomes during maturation would alter competence of the bovine oocyte for fertilization and subsequent development. Cumulus-oocyte complexes were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0–6 h or 16–22 h after initiation of maturation. Treatment with MG132 early in maturation prevented progression to meiosis II and reduced fertilization rate and the proportion of oocytes and cleaved embryos that became blastocysts. Conversely, treatment with MG132 late in maturation improved the percentage of oocytes and cleaved embryos that became blastocysts without affecting nuclear maturation or fertilization rate. Optimal results with MG132 were achieved at a concentration of 10 µM – effects were generally not observed at lower or higher concentrations. Using proteomic analysis, it was found that MG132 at the end of maturation increased relative expression of 6 proteins and decreased relative expression of 23. Among those increased by MG132 that are potentially important for oocyte competence are GAPDH, involved in glycolysis, TUBA1C, needed for organellar movement, and two proteins involved in protein folding (P4HB and HYOU1). MG132 decreased amounts of several proteins that exert anti-apoptotic actions including ASNS, HSP90B1, PDIA3 and VCP. Another protein decreased by MG132, CDK5, can lead to apoptosis if aberrantly activated and one protein increased by MG132, P4HB, is anti-apoptotic. Finally, the pregnancy rate of cows receiving embryos produced from oocytes treated with MG132 from 16–22 h of maturation was similar to that for control embryos, suggesting that use of MG132 for production of embryos in vitro does not cause a substantial decrease in embryo quality.

The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P < 0.05) level in SCNT oocytes that were treated post-fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos.