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

• Published in: Molecular reproduction and development 2014-04, Vol.81 (4), p.363-375
• Main Authors: Mayer, Sophia, Wrenzycki, Christine, Tomek, Wolfgang
• Format: Article
• Language: English
• Subjects: Animals; Carrier Proteins - antagonists & inhibitors; Carrier Proteins - physiology; Catalytic Domain - drug effects; Cattle; Cells, Cultured; Female; In Vitro Oocyte Maturation Techniques; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Meiosis - drug effects; Metaphase - drug effects; Multiprotein Complexes - drug effects; Naphthyridines - pharmacology; Oocytes - cytology; Oocytes - drug effects; Oocytes - enzymology; Oogenesis - drug effects; Phosphoproteins - antagonists & inhibitors; Phosphoproteins - physiology; Phosphorylation - drug effects; Phosphothreonine - analysis; Protein Kinase Inhibitors - pharmacology; Protein Processing, Post-Translational - drug effects; Protein-Serine-Threonine Kinases - antagonists & inhibitors; TOR Serine-Threonine Kinases - antagonists & inhibitors; TOR Serine-Threonine Kinases - drug effects
• ISSN: 1040-452X; 1098-2795
• DOI: 10.1002/mrd.22305

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.