Maturation Promoting Factor Kinase Activity Research Articles

259 Cdk7 AND CYCLIN H, BUT NOT Mat1, ARE INVOLVED IN MEIOTIC RESUMPTION OF PORCINE IMMATURE OOCYTE

The complex kinase Cdk-activating kinase (CAK) consists of the catalytic subunit Cdk7, regulatory subunit Cyclin H, and assembly factor Mat1. The CAK is essential for maturation-promoting factor (MPF) activation by phosphorylating threonine 161 (T161) of Cdc2 in mitosis. Although it is known that meiotic resumption of oocytes is regulated by MPF activity, the role of CAK in meiosis is still unclear. In the present study, we attempted to confirm the involvement of CAK in meiotic resumption of porcine immature oocyte. Cumulus–oocyte complexes (COC) were collected from antral follicles of gilts and cultured up to 48 h in TYH medium containing 20% porcine follicular fluid, 3.2 mg/mL of BSA, and 1.0 IU mL–1 of pregnant mare serum gonadotropin. The T161 phosphorylation level of Cdc2 in cultured oocytes was analysed by Western blot analysis. The transcripts were collected from noncultured or cultured oocytes, and Cdk7, Cyclin H, and Mat1 expression were detected by RT-PCR. Overexpression of Cdc2 or inhibition of Cdk7, Cyclin H, and Mat1 during oocyte maturation was performed by microinjection of mRNA or antisense RNA into ooplasm of immature COC and verified by Western blot or semiquantitative RT-PCR. Maturation-promoting factor kinase activity was assayed by Histone H1 kinase activity assay. Statistical analyses in this study were carried out by Student’s t-test. The T161 phosphorylation of Cdc2 was found during the culture period from 18 h to 48h, which was after germinal vesicle breakdown (GVB). Overexpression of Cdc2 increased the incidence of GVB at 18 h, but overexpression of mutant Cdc2 (replaced T161 by alanine) had no influence on GVB. These results indicate that T161 phosphorylation of Cdc2 is important for meiotic resumption. Next, we attempted to confirm the CAK function during oocyte maturation. Transcripts of Cdk7, Cyclin H, and Mat1 were detectable throughout the culture period. Inhibition of Cdk7 and Cyclin H caused a decrease in T161 phosphorylation and MPF activity, and the incidence of GVB was significantly lower than in nontreated oocytes. In contrast, Mat1-inhibited oocytes resumed meiosis and developed to the metaphase II stage, and the incidence was not different between Mat1-inhibited oocytes and nontreated oocytes. These results suggest that Cdk7 and Cyclin H are working as CAK and activate Cdc2 by T161 phosphorylation, although Mat1 is dispensable during oocyte maturation.

Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20

Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which we term endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.

320 MPF ACTIVITY AND DEVELOPMENTAL COMPETENCE IN DIFFERENT SIZES OF PREPUBERTAL GOAT OOCYTES

Developmental competence of prepubertal goat oocytes recovered from a slaughterhouse is low, probably due to an incomplete cytoplasmic maturation. Regulation of cytoplasmic maturation is still unknown, although maturation-promoting factor (MPF) is suggested to play an important role in this process. To better understand the role of MPF in cytoplasmic maturation, we have studied MPF kinase activity in oocytes with different developmental competence. Ovaries were obtained from a slaughterhouse, and oocytes were recovered by slicing and were selected according to morphological criteria. Some oocytes were denuded and classified in diameter groups (<110 μm, 110–125 μm, 125–135 μm, and >135 μm), placed in lysis buffer (50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 5 mM EDTA, 0.01% Brij35, 1 mM phenyl methyl sultonyl fluoride (PMSF), 0.05 mg/mL leupeptin, 50 mM 2-mercaptoethanol, 25 mM α-glycerophosphate, 1 mM Na-orthovanadate) and frozen in liquid N2. Cell extracts were stored at −80°C until use. The rest of oocytes were matured in vitro in medium TCM199 supplemented with hormones, 10% (DBS), and 400 μM cysteamine, for 27 h in 5% CO2 in air and 38.5°C. After IVM, a sample of oocytes were also denuded, classified by diameters, and frozen as described above. The rest of oocytes were used for IVF in mDM with spermatozoa capacitated with heparin and ionomicin. After 24 h, presumptive zygotes were cultured for 7 days in medium SOF in 5% CO2, 5% O2, and 90% N2 at 38.5°C. At 48 h post-insemination, we added 0.1 μL FBS per embryo. Embryonic development was evaluated with Hoechst staining after IVC. MPF kinase activity was detected using the MESACUP cdc2 kinase assay kit (MBL Woburn, MA, USA). Briefly, the oocyte extract corresponding to 10 oocytes was mixed with 10× reaction buffer (25 mM HEPES buffer (pH 7.5), 10 mM MgCl2) and 10% biotinylated MV peptide (SLYSSPGGAYC). We added 0.1 mM ATP to start the reaction. The mixture was incubated at 30°C for 30 min. The reaction was finished by adding 200 μL of PBS containing 50 mM EGTA. The phosphorylated MV peptide was detected by specific antibody using an ELISA procedure, and the OD was measured at 492 nm. Fisher's exact test was used to analyze IVC results, and ANOVA to analyze cdc2 kinase activity results. We considered differences statistically significant when P < 0.05. Results are shown in Table 1. We observed that embryonic cleavage and blastocyst rate increased with oocyte diameter. The MPF activity detected was higher in the largest oocytes after IVM. As a consequence, we could establish that oocytes with a higher MPF activity are more capable of maintaining embryonic development until the blastocyst stage, which may indicate the important role that MPF plays in cytoplasmic maturation. Table 1. Cleavage, blastocyst rate, and MPF kinase activity in different sizes of prepubertal goat oocytes

MAPK/ERK kinase (MEK) signalling is required for resumption of meiosis in cultured cumulus-enclosed pig oocytes.

Resumption of meiosis of mammalian oocytes is facilitated by the maturation promoting factor (MPF) and accompanied by activation of mitogen activated protein kinases (MAPK) which are phosphorylated by the MAPK kinase (MEK). In this study we examined the effects of PD 98059, which inhibits the activity of MEK, on in vitro maturation of pig oocytes. Cumulus-oocyte complexes (COCs) were cultured in the presence or absence of the drug (50 microM) for various time periods. To elucidate the influence of cumulus cells, COCs were first cultured in inhibitor-free medium, subsequently denuded, and incubated further in PD 98059 supplemented medium. Reversibility of drug action as tested following PD 98059 treatment of COCs by transferring them to drug-free medium. Culture of COCs in medium supplemented with PD 98059 prevents resumption of nuclear maturation in the majority of COCs. This inhibition was reversible and accompanied by a non-activation of both MAP and MPF. Addition of the MEK inhibitor to extracts of in vitro matured oocytes revealed that the kinase activities were not directly influenced by the inhibitor, suggesting a link between MAP and MPF kinases. Preincubation of COCs in inhibitor-free medium for 6 h followed by further culture of COCs or denuded oocytes in the presence of PD 98059 for various periods resulted in elevated MAP and MPF kinase activities, indicating an early and transient MEK signalling in the oocyte itself. These results support the idea that MAP and MPF are involved in the induction of germinal vesicle breakdown in porcine oocytes.

Biochemical characterization of the p34cdc2 protein kinase component of purified maturation-promoting factor from Xenopus eggs

Abstract Genetic studies in the fission yeast Schizosaccharomyces pombe and biochemical data in oocytes and eggs of Xenopus laevis have implicated the product of the cdc2+ gene as critical for the G2 to M transition in the cell cycle. The product of the cdc2+ gene is a 34-kDa serine/threonine protein kinase, designated p34cdc2, that is a component of purified maturation-promoting factor (MPF) and also of purified mammalian growth-associated histone H1 kinase. The biochemical properties of p34cdc2 H1 kinase activity in the MPF complex were studied. Phosphorylation of the p45cyclin component in the MPF complex by p34cdc2 exhibited kinetics consistent with an intramolecular reaction. On glycerol gradient centrifugation, MPF kinase against several substrates sedimented with an apparent Mr = 45,000-55,000. p34cdc2 was found to utilize ATP, GTP, and adenosine 5'-O-(3-thiotriphosphate) with apparent Km values of 75, 700, and 250 microM, respectively. The kinase activity was inhibited by beta-glycerophosphate, NaF, and zinc, whereas p-nitrophenyl phosphate was slightly stimulatory. The relative rates of phosphorylation of various substrates by MPF and growth-associated H1 kinase were similar. These findings should prove useful in further work on the regulation of MPF kinase activity and characterization of its substrates.