Dual Kinase/Phosphatase Regulating and Anchoring Protein Tribbles: Expression During Acquisition of Meiotic Competence and Meiosis in Mouse Oocytes.

Abstract

Mammalian oocytes arrested at prophase of meiosis I undergo a growth phase and acquisition of meiotic competence prior to resumption of meiosis and progression to metaphase of meiosis II. Reversible phosphorylation, involving kinases and phosphatases, is involved in both of these intra-oocyte processes. However, current understandings of how numerous reversible phosphorylation events are orchestrated during acquisition of meiotic competence and maturation are significantly lacking. The Tribbles protein was originally described as a cell cycle regulator required for mesendodermal differentiation and oocyte specification during Drosophila embryogenesis. Drosophila Tribbles protein regulates cell proliferation by promoting degradation of CDC25 protein phosphatase. Mammalian somatic cells contain three Tribbles genes (Tribbles 1, 2, and 3) with more diverse functions, such as anchoring proteins that regulate protein kinase B, mitogen-activated protein kinases and CCAAT/enhancer-binding protein alpha. The objectives of this study were to determine if mammalian oocytes expressed Tribbles and compare Tribbles mRNA levels during acquisition of meiotic competence and maturation. Oocytes were collected from CF1 mice at postnatal d10-13 [growing / germinal vesicle-intact (GV-intact) / meiotically incompetent (Incomp)] and d19-21 following gonadotropin stimulation [GV-intact / meiotically competent (Comp)]. Denuded GV-intact oocytes were pooled and used for mRNA isolation or GV-intact/Comp oocytes were placed into culture and matured for 2, 7, and 16hrs to germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII), respectively, prior to mRNA isolation. Gene expression for Tribbles 1, 2, 3, and actin at each stage of oocyte development were quantified by real-time PCR, in triplicate per experiment, in three separate experiments. Expression ratios and/or fold changes were tested for significance by a pairwise fixed reallocation randomization test, and p<0.05 was considered significantly different. Mouse oocytes at all stages assessed expressed all three Tribbles genes. As oocytes gained meiotic competence gene expression of Tribbles 1 and 3 did not significantly change. However, during the transition from GV-intact Incomp to Comp Tribbles 2 gene expression significantly increased 15.7 fold; p<0.001. Tribbles 1 mRNA levels also did not significantly change during transitions from GV-intact to GVBD, to MI, or to MII. During the GV-intact to GVBD transition Tribbles 2 and Tribbles 3 mRNA levels significantly decreased (4.4 fold; p<0.01) and increased (5.7 fold; p<0.01), respectively. Finally, while no significant changes of any Tribbles mRNA occurred during the transition through MI, Tribbles 2 mRNA was found to significantly increase (2.1 fold; p<0.01) during the MI to MII transition. In conclusion, this is the first report of mammalian oocytes expressing all three Tribbles, which are known dual kinase/phosphatase regulatory and anchoring proteins. Tribbles 2 gene expression and mRNA levels change most significantly during acquisition of meiotic competence and meiotic maturation. Continued studies are required at the protein and functional levels and will help elucidate regulatory pathways of meiotic competence acquisition, meiotic maturation, and attainment of embryonic developmental competence. (poster)