Inactivation of mTor: A Tool to Investigate Meiotic Progression and Translational Control During Bovine Oocyte Maturation

1BVN Neustadt/Aisch, 2Faculty of Veterinary Medicine, Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals, Justus-Liebig-University Giessen, 3Reproductive Cell Biology Unit, Leibniz Institute for Farm Animal Biology, 4Reproductive Biochemistry Unit, Leibniz Institute for Farm Animal Biology
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Abstract

Although routinely used in breeding programs, in vitro maturation (IVM) of bovine oocytes and in vitro production (IVP) of embryos are nevertheless still the subject of basic research owing to suboptimal IVM conditions and variations in the developmental competence of the starting oocytes. In the present study we provide a method to inhibit the Ser/Thr kinase mTor during IVM using two independent inhibitors, Torin2 and Rapamycin. Both substances have different effects on meiotic progression and translational control and may allow discrimination between the mTorC1 and mTorC2 complex functions. The effects of the inhibitors are monitored by inspection of the chromatin configuration using aceto-orcein-staining as well as Western blotting and immunohistochemical analysis of the phosphorylation state of the translational repressor 4E-BP1, which is a prominent mTor target. Whereas Torin2 arrests bovine oocytes in the M I stage and inhibits 4E-BP1 phosphorylation, Rapamycin inhibits asymmetric division and does not influence 4E-BP1. Investigations utilizing these reactions can provide deeper insights into the regulatory events involved in meiotic maturation. Moreover, special focus can be placed on the temporal and spatial regulation of translational control. Such findings can contribute to the definition of the developmental competence of oocytes and to an improvement of IVM conditions.