The inhibition of cysteine peptidases improves in vitro maturation of porcine cumulus-oocyte-complexes
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The cell permeable cysteine peptidase inhibitor, E-64, has shown utility as a potent and irreversible inhibitor for many cysteine proteases. Consequently, it has been studied in several model systems, including ones involving oocyte and embryo culture. In cattle, administration of E-64 during oocyte IVM or embryo culture resulted in improvements in both oocyte maturation and embryo development. In this study the effects of E-64 were investigated in a porcine IVM/IVF system. Significantly increased rates of oocyte maturation were recorded for COCs treated with E-64. In addition, an increase in cleavage rates was found. However, blastocyst development was not altered. Furthermore, in vitro maturation of porcine COCs using E-64 was performed with COCs segregated into morphologically distinct developmental groups. Those COCs defined as 'good' matured in significantly higher proportions when E-64 was added to the in vitro maturation medium compared to 'good' COCs in the absence of E-64. In addition, 'good' COCs treated with E-64 produced blastocysts with numerically higher total cell counts. In vitro matured COC and oocyte mRNA was extracted and compared to in vivo derived oocyte mRNA. There was a significant difference in cathepsin K mRNA abundance between in vitro matured oocytes and in vivo produced oocytes. However, there was no significant difference in cathepsin B mRNA between the samples, despite that such a difference had been expected based on previous reports in bovine oocytes. In vitro maturation with cathepsin B and cathepsin K specific inhibitors produced oocytes that reached MII at percentages consistent with those of untreated oocytes. Protein expression was used to determine if cathepsin B was being affected at the protein level. Western blots revealed cathepsin B in COCs, but not in denuded oocytes. Additionally, immunocytochemistry verified these results by showing cathepsin B localization in cumulus cells but not in the denuded oocyte.
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