Abstract
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? h or 16?2 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 mM ?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 antiapoptotic 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?2 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.
Citation: You J, Lee E, Bonilla L, Francis J, Koh J, et al. (2012) Treatment with the Proteasome Inhibitor MG132 during the End of Oocyte Maturation Improves Oocyte Competence for Development after Fertilization in Cattle. PLoS ONE 7(11): e48613. doi:10.1371/journal.pone.0048613 Editor: Shree Ram Singh, National Cancer Institute, United States of America Received July 12, 2012; Accepted September 27, 2012; Published November 7, 2012 Copyright: ?2012 You et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2010-85122-20623 from the USDA National Institute of Food and Agriculture. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Co-author, Jeremy Block, is affiliated with a company called Ovatech. He has no other relationship with any patents, products, etc. There are no relevant declarations relating to employment, consultancy, patents, products in development or marketed products. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Introduction
The proteasome, a multisubunit proteolytic complex involved in degradation of ubiquitinated proteins, plays a crucial role in assuring completion of meiosis and formation of a developmentally-competent embryo. Early in maturation, completion of meiosis I requires inactivation of maturation promoting factor (MPF) through a process mediated by proteasomal cleavage of ubiquitinated cyclin B1 [1]. Other aspects of oocyte function during maturation are also under control of the proteasome. In mice, for example, the proteasome is required for the initiation and maintenance of translation of mRNA for the RNA binding protein SLBP [2]. Abundance of another protein involved in RNA processing, CPEB, is under negative regulation by proteasomes in Xenopus [3]. In addition, cumulus cells encasing the oocyte require proteasomal activity for optimal function as indicated by negative effects of the proteasomal inhibitor MG132 on progesterone production and expression of genes involved in expansion of the extracellular matrix [4]. This peptide aldehyde, N-(benzyloxycarbonyl)leucinylleucinylleucinal, functions as a substrate analog and transition-state inhibitor of the chymotrypsin-like activity of the proteasome [5]. Late in the process of oocyte maturation, the proteasome may contribute to a reduction in the functional properties of the oocyte. Treatment with MG132 reduced the effect of in vitro aging on oocyte competence in the mouse [6]. Furthermore, treatment of oocytes with MG132 late in maturation increased abundance of specific transcripts and improved developmental competence of parthenogenetically-activated oocytes in the pig [7]. If inhibition of the ubiquitin-proteasome pathway late in maturation improves oocyte competence, it may be possible to improve the success rate of assisted reproductive technologies that utilize in vitro matured oocytes. The purpose of the present series of experiments was to test the hypothesis that treatment of bovine oocytes with MG132 at the end of maturation would improve developmental competence of the oocytes and resultant embryos while addition of MG132 at the beginning of maturation would reduce oocyte competence. An additional goal was to assess specific proteins whose relative abundance in the oocyte was altered by MG132 late in maturation with the goal of identifying candidate molecules responsible for actions of MG132 on oocyte competence.
Materials and Methods
Use of animals was approved by the University of Florida Institutional Animal Care and Use Committee.
