Inhibition of Cysteine Proteinases During In Vitro Oocyte Maturation Improves Development of Porcine Cumulus-Oocyte-Complexes.

Abstract

Biochemical differences are known to exist between oocytes that give rise to viable blastocysts compared to oocytes that give rise to embryos that are developmentally compromised. Indeed, mRNA for specific proteolytic enzymes (e.g. cathepsin B) have been shown to be more abundant in in vitro matured bovine oocytes that have diminished developmental potential. The effects of the cysteine proteinase inhibitor, E-64, was recently investigated in bovine cumulus-oocyte-complexes (COCs) chosen to represent both poor- and good-quality oocytes. These reports revealed that the addition of E-64 promoted both oocyte maturation and subsequent embryo development, presumably through inhibition of cathepsin B. We sought to determine if similar results would be obtained in a different oocyte/embryo culture system (i.e. for porcine oocytes). Specifically, it was hypothesized that E-64 would promote maturation of lower-quality oocytes within an aspirated pool; E-64 was also predicted to have little influence on normal oocytes. E-64 was added to the maturation media of ‘good’ v. ‘poor’ COCs that were segregated based on morphological characteristics of the oocytes and by the extent of cumulus cell development. To further determine if cathepsin B itself was influencing COCs, the cathepsin B specific inhibitor, CA-074, was also used during in vitro maturation (IVM) of porcine oocytes. In addition, an analysis of cathepsin mRNA abundance was also performed on cDNA obtained from in vitro matured MII oocytes as a model for ‘lower’ quality oocytes and in vivo (IVV) MII oocytes as a model for ‘good’ quality oocytes. A alterations in cathepsin mRNA abundance were also investigated in IVM porcine COCs and denuded oocytes treated with E-64. Addition of E-64 to IVM medium increased the frequency of maturation in MII oocytes. The highest maturation percentages (74%) were seen in treatment groups containing 20μM E-64 compared to those incubated in the presence of 10 μM E-64 (74% v. 53%; P<0.05) or without E-64 (55%; P<0.05; N=1,750 total oocytes). The percent maturation of ‘good’ oocytes cultured in E-64 was significantly higher than ‘good’ oocytes not exposed to E-64 (68% v. 56%; P<0.001). ‘Poor’ oocytes cultured with E-64 also had significantly higher maturation percentages compared to ‘poor’ oocytes cultured without E-64 (43% v. 31%; P<0.0001). Day six blastocysts that arose from E-64 treated IVM oocytes exhibited no significant increase in total cell numbers relative to untreated ones (34.9 v. 29.8; P=0.19). The use of a cathepsin B specific inhibitor, CA-074, during IVM of oocytes showed no significant effect on oocyte maturation. As with previous experiments, the addition of 20μM E-64 increased oocyte maturation percentages (68% v. 50%; P<0.05). Real-time PCR analysis revealed that cathepsin K mRNA was elevated in IVM MII oocytes compared to IVV MII oocytes as well as in IVM COCs in relation to their denuded counterparts. Cathepsins B and L were not significantly different between IVM and IVV MII oocytes, however cathepsin B did show an increase in abundance in IVM MII COCs compared to IVM denuded MII oocytes. These data suggest that improvements in porcine oocyte maturation arising from E-64 treatment may be due to inhibition of multiple cysteine peptidases and not just from the suppression of cathepsin B, as has been suggested in bovine oocytes. Differences between treatments were determined by SAS with post-test by Tukey's Multiple Comparison Test.