When the nucleus in G0/G1 phase is transferred to an enucleated oocyte by nuclear transfer (NT), its nuclear envelope is broken, followed by condensation of chromosome structure by maturation promoting factor (MPF). This morphological remodeling of the transferred interphase nucleus seems to be essential for subsequent development of NT embryos. In this study, we treated porcine NT embryos with caffeine, which has been reported to increase MPF activity, to keep their MPF level high during NT. When 2.5 mM caffeine was added to the handling medium, the proportion of NT embryos showing condensed chromosome increased significantly (P < 0.05). In NT embryos treated with caffeine, the activity of p34cdc2 kinase was significantly (P < 0.05) higher than in those without caffeine at 3 h post-injection. In addition, the rate of development to the blastocyst stage after activation was significantly (P < 0.05) higher in NT embryos treated with caffeine. These results indicate that caffeine treatment can increase not only the rate of chromosome condensation but also the developmental rate to the blastocyst stage of porcine NT embryos. This action is most likely due to the support/increase of MPF activity throughout the process of NT.
Manabu Kawahara, Takuya Wakai, Ken-Ichi Yamanaka, Jin Kobayashi, Satoshi Sugimura, Takashi Shimizu, Hiromichi Matsumoto, Jin-Hoi Kim, Hiroshi Sasada and Eimei Sato
Hafiza Khatun, Yasuhiko Wada, Toshihiro Konno, Hideki Tatemoto and Ken-ichi Yamanaka
We have previously reported that regulation of endoplasmic reticulum (ER) stress during in vitro culture acutely increases bovine embryo developmental rate and cryotolerance; these data indicate that ER stress is a critical factor reducing the quality of in vitro-produced embryos. In the current follow-up study, we examined whether ER stress attenuation during in vitro maturation influences meiotic maturation, oocyte quality, and subsequent embryonic development. Bovine cumulus oocyte complexes (COCs) derived from slaughterhouse ovaries were matured with or without tauroursodeoxycholic acid (TUDCA), a selective inhibitor of ER stress (0, 50, 100, and 200 µM) for 22 h followed by in vitro fertilization, and zygotes were cultured for 8 days. Of the different doses of TUDCA, 100 μM TUDCA significantly increased the maturation rate, and decreased reactive oxygen species in denuded oocytes, and appeared lower number of apoptotic cells in matured COCs. Subsequently, treatment of TUDCA (100 µM) decreased the localization and amount of GRP78/BIP protein level as well as ER stress (GRP78/BIP, PERK, IER1, ATF4, and XBP1) and apoptosis (CHOP and BAX)-related gene expression, while it increased the anti-apoptotic gene BCL2 level in matured COCs. Moreover, addition of TUDCA (100 µM) during IVM significantly improved the blastocyst formation rate (43.6 ± 1.8% vs 49.7 ± 1.3%) and decreased the number of apoptotic cells (7.7 ± 1.1% vs 5.03 ± 0.6%) in blastocysts. These findings suggest that the presence of ER stress during maturation impairs the developmental competence of bovine COCs and that this process can be reversed by TUDCA.