Inactivation of mTor arrests bovine oocytes in the metaphase-I stage, despite reversible inhibition of 4E-BP1 phosphorylation

SUMMARY The mammalian target of rapamycin (mTor), a Ser/Thr protein kinase, is implicated in the phosphorylation-triggered inactivation of translation repressors, the so-called eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Previous observations in porcine and bovine oocytes revealed an increasing phosphorylation of 4E-BP1 during meiotic maturation. This factor is hypophosphorylated in the germinal-vesicle (GV) stage and its phosphorylation peaks in the metaphase II (M II) stage. In the present approach we intended to block 4E-BP1 phosphorylation specifically to impair initiation of translation and elucidate effects on resumption of meiosis. Torin2, which acts as an active-site mTor inhibitor, reduces 4E-BP1 phosphorylation without any effect on eIF4E and arrests up to 60% of the oocytes in the M I stage. Effects of Torin2 treatment, analyzed by site-specific substrate phosphorylation, were also observed at protein kinase B (Akt or PKB), and cyclin dependent kinases (CDKs). Only minor side effects were found at protein kinase A, C (PKA, PKC), ATM/ATR (Ataxia telangiectasia mutated/AT and Rad3-related protein), and the mitogen activated protein kinases (MAPK) ERK1,2. The inhibition of 4E-BP1 phosphorylation by Torin2 is reversible when cultivating oocytes for additional 24 hr in Torin2-free medium. Even so, oocytes persist in the M I stage. This may indicate the necessity of spatiotemporally regulated translation during meiosis, which cannot be restored later. In conclusion, Torin2 enables an effective and specific inhibition of 4E-BP1 phosphorylation, which may be valuable to investigate maturation specific protein synthesis in more detail. Mol. Reprod. Dev. 81: 363–375, 2014. © 2014 Wiley Periodicals, Inc.