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

Fully grown bovine oocytes (arrested at prophase I; germinal vesicle -GV- stage) resume meiosis spontaneously when they are released from their follicles and transferred to a suitable culture medium. Previous investigations using the application of different inhibitors to in vitro culture media^{1, 2} revealed that activation of protein kinases and de novo protein synthesis trigger the maturation of mammalian oocytes and arrest them in metaphase II (M II), the stage suitable for fertilization. In the present study we describe a method to inhibit the Ser/Thr kinase mTorduring IVM of bovine oocytes. This approach might provide deeper insights into the complex processes involved in the regulation of protein synthesis in the context of meiotic maturation (transition from GV-stage to M II), because mTor links the phosphorylation of specific factors directly to translational control^{3, 4}.

The focus on the investigation of translational control reflects the importance of this process; fully grown oocytes are transcriptionally silent and protein synthesis relies on the activation of stored, dormant mRNAs⁵. In this context, mTor plays a predominant role. The kinase directly phosphorylates and inactivates repressors of the mRNA cap-binding protein eIF4E, the so-called 4E-binding proteins (4E-BP1-3), and thereby allows the formation of the 5´-mRNA-cap binding complex eIF4F (composed of eIF4E, the scaffold protein eIF4G and the RNA helicase eIF4A). Together with other factors it also stimulates ribosome binding and translation initiation⁶.

mTor, however, exists as two complexes: mTorC1 and mTorC2. Each complex is composed of different major regulators, differs in sensitivity to Rapamycin and has different cellular targets⁷. The major regulator of mTorC1, Raptor (regulatory-associated protein of mTOR), phosphorylates components of the translational machinery, namely ribosomal proteins (for instance RPS6 at Ser235/36) and the translational repressor 4E-BP1 (at Thr37/46/65/70). The major regulator of mTorC2, Rictor (Rapamycin-insensitive companion of mTOR), is Rapamycin- resistant and phosphorylates Akt (PKB) which in turn phosphorylates mTorC1. Preliminary investigations in bovine oocytes during IVM revealed different transient activities of mTorC1 and mTorC2 during IVM. In the GV-stage of oocytes the mTorC2 is active³ ; it is inactivated in the course of IVM. In contrast, mTorC1 shows the opposite behavior³. These results correspond with findings showing that 4E-BP1 phosphorylation is lower in the GV-stage, continuously increases during IVM, and is highest in the M II stage^{8, 9}.

However, mTorC1 and C2 both respond to the active site inhibitor Torin2¹⁰ and might have other (yet unknown) targets. Candidates are meiotic spindle-forming or regulatory proteins, since mTor associates with meiotic spindles during chromatin segregation.

From a practical point of view, it should be noted that in vitro systems yield only 30-40 % transferable embryos in the bovine species ¹¹. The causes for this could be suboptimal in vitro conditions and/or differences in the developmental competence of the starting oocytes which occur despite their selection from follicles of a defined size. However, detailed investigations of meiotic maturation on a molecular level can contribute to the optimization of IVM systems. Furthermore, oocytes might be selected according to their developmental competence, for instance by IVM systems under inhibitory conditions (see discussion). Hence, in the procedure presented here, we used two independent mTor inhibitors, Torin2 and Rapamycin, which resulted in different chromatin statuses and differential phosphorylation of 4E-BP1. Interestingly, approximately 20 % of the oocytes overcame the Torin2 block and might thus be candidates which possess a high developmental competence.