Dynamics of protein phosphorylation during meiotic maturation

Abstract

Purpose To ask whether distinct kinase signaling pathways mediate cytoplasmic or nuclear maturation of mouse oocytes and if in vitro maturation influences the distribution and timing of these phosphorylation events. Methods Mouse cumulus oocyte complexes (COCs) were matured under conditions known to influence oocyte quality (basal or supplemented media) and assayed with epitope specific antibodies that would distinguish between Cdk1 or tyrosine kinase targets at 0, 2, 4, 8, and 16 hrs. Semi-quantitative image analysis was used to assess the topographical patterns of protein phosphorylation during in vitro maturation. In vitro fertization and embryo culture were used to examine the effects of culture conditions on developmental potential. Results Protein tyrosine phosphorylation increased during meiotic progression from methaphase-I to metaphase-II. Levels were significantly higher in the oocyte cortex. Levels of cortical staining are enhanced in oocytes matured in supplemented media that displayed higher developmental competence. In contrast, bulk substrates for Cdk1 kinase localize to the meiotic spindle while cytoplasmic levels of kinase activity increase throughout meiotic progression; culture media had no measurable effect. Ablation of the tyrosine kinase Fyn significantly reduced cortical levels of tyrosine phosphorylation. Conclusions The findings indicate that distinct signaling pathways mediate nuclear and cytoplasmic maturation during in vitro maturation in a fashion consistent with a role for tyrosine kinases in cortical maturation and oocyte quality.

The Effect of Cilostamide on Gap Junction Communication Dynamics, Chromatin Remodeling, and Competence Acquisition in Pig Oocytes Following Parthenogenetic Activation and Nuclear Transfer1

“…This antibody is known to detect epitopes of many relevant substrates for M-phase specific kinases and is a specific biomarker for the G2/M cell cycle transition [39][40][41][42][43].…”

“…Oocytes were simultaneously fixed and extracted in a microtubule-stabilizing buffer containing proteases and phosphatases inhibitors, as described elsewhere [40,42,43] and then incubated overnight with primary antibody at 4°C. The primary antibodies used were rabbit polyclonal anti-phospho-Histone H3 (Ser28) (diluition 1:100, Merk Millipore, Temecula, CA, USA) or mouse monoclonal anti-phospho-Ser/Thr-Pro antibody, MPM2 (dilution 1:100; Merk Milliopore, Temecula, CA, USA).…”

“…To further confirm that SC and GVII-IV configurations were different, and more precisely that GVII-IV are typical configurations that occur between removal of the COC from the follicle (i.e when spontaneous meiotic resumption occur) and GVBD, we conducted immunofluorescent staining of H3 Ser 28 and MPM2, at the time of collection and after 24h of culture, as these have been indicated as markers of meiotic progression [32,[39][40][41][42][43]49]. In our conditions, H3 started to be phosphorylated at Pro-MI, while no differences were observed between SC, GVI and GVII-IV stages (Fig.…”

The impact of vitrification on immature oocyte cell cycle and cytoskeletal integrity in a rat model

“…Oocytes were fixed at 37°C for 5 min in 3 % paraformaldehyde then 30 min at 37°C in a microtubule stabilizing buffer extraction fixative (MTSB XF) (100 mM PIPES, 5 mM MgCl2, 2.5 mM EGTA, 2 % formaldehyde, 0.1 % Triton-X-100, 1 mM taxol, 10 U/ mL aprotinin, 50 % deuterium oxide) with phosphatase inhibitors (40 uM phenylarsine oxide, 100 uM NaVO 4, and 50 ng/ml Calyculin A) [9]. All COCs were labeled with M-phase specific antibody (MPM-2) to establish meiosis re-entry kinetics for thawed COCs based on the transition from nuclear to cytoplasmic protein phosphorylation, and counterstained with Hoechst 33258 (1 ug/ ml).…”

“…All COCs were labeled with M-phase specific antibody (MPM-2) to establish meiosis re-entry kinetics for thawed COCs based on the transition from nuclear to cytoplasmic protein phosphorylation, and counterstained with Hoechst 33258 (1 ug/ ml). All COCs were labeled with 1 U/ml Alexafluor 568 phalloidin (Invitrogen) to monitor alterations in f-actin and triple labeled samples were imaged as described previously [9]. 3D data sets were collected on a Zeiss LSM 510 confocal microscope using excitation and emission settings such that chromatin configuration (Hoechst 33258) could be directly compared to cell cycle status (MPM-2) and filamentous-actin (F-actin, Alexafluor 548-phalloidin) of individual oocytes.…”