Optimization of parthenogenetic activation protocol in porcine

The effects of the electrical field strengths, number of pulses, and post-activation media on chromatin conformation and parthenogenetic development were studied to optimize the activation protocol for porcine nuclear transfer. In experiment 1, electrical field strengths were examined. Oocytes were subjected to square direct current pulses at output voltages of 1.2, 1.7, 2.2, and 2.7 kV/cm for 1 x 30 microsec. The voltage resulting from experiment 1 was 2.2 kV/cm, in which 50.0% of activated oocytes developed to blastocysts in vitro. In experiment 2, the influence of 1, 2, and 3 pulses on blastocyst development was tested using field strengths and post-activation medium described in experiment 1. Oocytes activated by a single 30 microsec pulse of 2.2 kV/cm DC yielded a higher blastocyst rate (56.3%) than oocytes activated by 2 or 3 pulses (<42.5%). In experiment 3 and 4, we investigated the effects of cytochalasin B (CB), cycloheximide (CH), and CB + CH on nuclear development stages and parthenogenetic development following a single 30 microsec pulse of 2.2 kV/cm DC. The percentage of activated oocytes was not different among CB (93.3%), CB + CH (98.3%), control (80.0%), and CH (80.0%) groups 12 hr after activation. Treatment with CB (57.5%) or CB + CH (53.8%) enhanced the blastocyst rate compared with other groups, CH (23.8%) treated- and control group (18.8%). The results demonstrated that a single 30 microsec pulse of 2.2 kV/cm DC followed by culturing in post-activation medium with CB for 5 hr were effective parameters for parthenogenetic activation and blastocyst formation of in vitro matured porcine oocytes which suggests that a single calcium rise is sufficient to activate pig oocytes and to achieve high rate of blastocyst development.     

Effect of 6-dimethylaminopurine on electrically activated in vitro matured porcine oocytes

The effect of the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the maturation promoting factor (MPF) activity, pronuclear formation, and parthenogenetic development of electrically activated in vitro matured (IVM) porcine oocytes was investigated. Oocytes were activated by exposure to two DC pulses, each of 1.5 kV/cm field strength and 60 microsec duration, applied 1 sec apart. In the first experiment, subsequent incubation with 2 or 5 mM 6-DMAP for 3 hr increased the incidence of blastocyst formation compared with no treatment, whereas incubation with 2 or 5 mM 6-DMAP for 5 hr did not. In the proceeding experiments, oocytes exposed to 6-DMAP were incubated with 2 mM of the reagent for 3 hr. Assaying histone H1 kinase activity in the second experiment revealed that the levels of active MPF in electrically activated oocytes treated with 6-DMAP were depleted more rapidly and remained depleted for longer compared with electrical activation alone. The kinetics of MPF activity following 6-DMAP treatment were similar to that found in inseminated oocytes in the third experiment. The effect of 6-DMAP was correlated with an increased incidence of parthenogenetic blastocyst formation. A fourth experiment was undertaken to examine the diploidizing effect of 6-DMAP. Electrically activated oocytes treated with 6-DMAP and cytochalasin B, either alone or in combination, displayed a higher incidence of second polar body retention compared with those that were untreated or treated with cycloheximide alone. After 6 days of culture in vitro, parthenotes exposed to 6-DMAP, either alone or in combination with cytochalasin B, formed blastocysts at a greater rate compared with those exposed to cytochalasin B alone, cycloheximide alone or no treatment. The combined 6-DMAP and cytochalasin B treatment induced the highest rate of blastocyst formation (47%), but the numbers of trophectoderm and total cells in these blastocysts were lower compared with those obtained following exposure to 6-DMAP alone. These results suggest that the increased developmental potential of 6-DMAP-treated parthenotes may be attributable to the MPF-inactivating effect of 6-DMAP, rather than the diploidizing effect of 6-DMAP.     

Kinetics of meiotic progression, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes: species specific differences in the length of the meiotic stages

The kinetics of nuclear maturation, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes were examined. A further objective was to determine the duration of the meiotic stages during the maturation process. Porcine and bovine cumulus-oocyte complexes (COCs) were incubated in TCM 199 supplemented with 20% (v/v) heat inactivated fetal calf serum (FCS), 0.05microg/ml gentamycin, 0.02mg/ml insulin, 2.5microg/ml FSH and 5microg/ml LH. COCs were removed from the culture media in hourly intervals starting immediately after recovery from the follicle until 24 (bovine) or 48h (porcine) of culture. Oocytes were either fixed to evaluate the maturation status or the activity of MPF, assessed by its histone H1 kinase activity, and MAP kinase were determined by a radioactive assay simultaneously. In oocytes of both species, the MPF activity oscillated during the culture period with two maxima corresponding with the two metaphases: between 27-32 and after 46h (porcine) and between 6-9 and after 22h (bovine). There was a temporary decline in activity after 33-38 (porcine) and after 19h (bovine), which corresponded with anaphase I and telophase I. MAP kinase activity increased during the whole culture period and reached maximum levels after 47 (porcine) and after 22h (bovine). In porcine oocytes, the MAP kinase was activated before GVBD and MPF activation. In bovine oocytes, MPF and MAP kinase were activated at approximately the same time as the GVBD (8-9h of incubation). In average porcine, oocytes remain 23.4h in the germinal vesicle (GV) stage (13h in GV I, 5.7h in GV II, 3.2h in GV III and 1.5h in GV IV), 0.9h in diakinese, 9.6h in the metaphase I, 2.8h in anaphase I and 1.9h in telophase I of the first meiotic division. In bovine oocytes, the temporal distribution of the meiotic stages were 8.5h for the GV stage, 1.2h for diakinese, 8.3h for metaphase I, 1.6h for anaphase I and 1.9h for telophase I. These results indicate that the duration of the meiotic stages differs between the species and that MAP kinase is activated before MPF and GVBD in porcine oocytes.     

Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation

The present study investigated the effect of lactate/pyruvate supplement in culture medium and of chemical activation after electric stimulus on in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. In vitro matured gilt oocytes were enucleated, reconstructed with fetal fibroblasts, and simultaneously fused/activated using a single pulse of 2.0 kV/cm for 30 microsec. In Experiment 1, reconstructed embryos were cultured in North Carolina State University (NCSU)-23 medium supplemented with either 5.5 mM glucose (Group A) or lactate (5.0 mM)/pyruvate (0.5 mM) (Group B). Compared to Group A, cleavage rate (64% vs. 47%) was higher and more blastocysts developed in Group B (17% vs. 6% at Day 6, 21% vs.11% at Day 7). Experiment 2, embryos reconstructed by electric stimulus (2.0 kV/cm for 30 microsec) were subjected to three activation protocols: (1) no chemical activation (Group C), (2) 7.5 microg/ml cytochalasin B treatment at 2 hr after electric stimulus (Group D), and (3) 5 microg/ml 6-dimethylaminopurine (Group E) treatment at 2 hr after electric stimulus. The reconstructed embryos were cultured for 7 days in NCSU-23 medium supplemented with lactate (5.0 mM)/pyruvate (0.5 mM). The rates of blastocyst formation on Day 6 and Day 7 in Group C (17 and 20%, respectively) or Group D (15, 20%, respectively) were higher than in Group E (9 and 12%, respectively). The percentage of two pseudo-pronucleus (PPN) formations in Group D (88%) was significantly higher than in Group C (71%) and Group E (72%). Mean cell numbers of blastocysts in Group D (63.4 +/- 15.8) were higher than in Group C (43.9 +/- 16.5) and Group E (32.9 +/- 17.9), due to increased trophectoderm (TE) cell numbers. Our results indicate that supplementing NCSU-23 medium with lactate/pyruvate and exposure of cytochalasin B after electrical stimulus can improve in vitro developmental competence of porcine SCNT embryos.     

Parthenogenetic activation of porcine oocytes by electric pulse and/or butyrolactone I treatment

The goal of the present research was to study the parthenogenetic activation of porcine oocytes following treatment with the specific cyclin-dependent kinase inhibitor butyrolactone I (BL I). In Experiment I, the effective dose of BL I was determined by the rates of the subsequent pronuclear formation in oocytes after the activation. In Experiment II, BL I was further tested alone or in combination with an electric pulse. The efficiency of the various treatments to induce activation and parthenogenetic development was examined. In Experiment III parthenogenetic development of activated oocytes in two different media was compared. Cleavage and blastocyst developmental rates were examined, and number of cells in the blastocysts was determined. Our results indicate that, in pig, the optimal activation dose for BL I was 150 microM; a combined electrical and BL I treatment resulted in superior cleavage rates compared to an electric pulse, 150 microM of BL I, or 200 microM of BL I alone (74%, 60%, 41%, and 42%, respectively; P < 0.05); and the rate of parthenogenetic development of activated oocytes to the blastocyst stage in mNCSU37 medium was significantly higher than that in Whitten's medium (59% vs. 5%, P < 0.05) and the resulting day-6 blastocysts had higher cell numbers (35.5 +/- 14.1 vs. 19.5 +/- 2.5). This activation protocol might be useful in porcine nuclear transfer experiments and for the generation of parthenogenetic fetuses.     

Mitogen-activated protein kinase (MAP kinase) activation in Xenopus oocytes: Roles of MPF and protein synthesis

Mitogen-activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone-induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase ll oocytes under two different forms: an inactive 41-kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42-kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels. During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M-phase promoting factor (MPF), a complex between the p34^cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase l and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s). © 1993 Wiley-Liss, Inc.     

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified.     

Timing of Plk1 and MPF activation during porcine oocyte maturation

A Polo-like kinase 1 (Plk1) appears involved in an autocatalytic loop between CDC25C phosphatase and M phase promoting factor (MPF) in Xenopus oocytes and leads to activation of MPF that is required for germinal vesicle breakdown (GVBD). Although similar evidence for such a role of Plk1 in MPF activation during maturation of mammalian oocytes is absent, changes in Plk1 enzyme activity correlate with MPF activation, Plk1 co-localizes with MPF, and microinjection of antibodies neutralizing Plk1 delays GVBD. In this study, we exploited the prolonged time required for maturation of porcine oocytes to define precisely the timing of Plk1 and MPF activation during maturation. GVBD typically occurs between 24 and 26 hr of culture in vitro and meiotic maturation is completed after 40-44-hr culture. We find that Plk1 is activated before MPF, which is consistent with its role in activating MPF in mammalian oocytes.     

Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro.

Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.     

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.     

Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.

The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone.     

Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes.

The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation.     

Parthenogenetic activation and subsequent development of rat oocytes in vitro.

Studies were undertaken to determine whether electrical stimulation, or ethanol treatment alone or in combination with 6-dimethylaminopurine (6-DMAP) influenced the rate of parthenogenetic activation of rat oocytes. The percentages of activated oocytes with pronuclei (89-91%) and those developed to the two-cell stage (68-72%) were significantly higher after electrical stimulation with direct current (DC) at 100 V/mm, 99 microsec once or twice, than when other DC voltages (75, 150, and 200) were applied or when ethanol or 6-DMAP treatment was given alone. However, none of the activated oocytes developed beyond the four-cell stage. The percentages of activated oocytes with pronuclei (100%) that developed to the two-cell (100%), eight-cell (89%) and blastocyst stages (50%) were significantly higher when electrical stimulation was followed by treatment with 2 mM 6-DMAP for 4 hr than when other combined procedures were applied. In conclusion, the results of the present study clearly showed that combined treatment of electrical stimulation or ethanol with 6-DMAP induces parthenogenetic activation and subsequent development of rat oocytes in vitro.     

Effects of spindle removal on MPF and MAP kinase activities in porcine matured oocytes

Intracellular localization of maturation/M-phase promoting factor (MPF) and mitogen activated protein (MAP) kinase in mature oocytes has been examined by immunocytochemical methods and the authors of these studies have reported that they are localized on spindles during M-phase. Although these reports showed the relative localization of MPF and MAPK on spindles, it has never been shown whether these kinases are present in the cytoplasm and, if they are present, how many parts of the kinases are localized on the metaphase spindle. In the present study, we made quantitative analyses of MPF and MAP kinase localized on oocyte spindles by kinase assays and immunoblotting after removal of the spindles from porcine mature oocytes. First, we certified their intracellular distribution by immunocytochemical methods and observed sharp signals of cyclin B1 on spindle poles and MAP kinase signals on the microtubule of metaphase spindles. In contrast to these results by immunostaining, the amounts of MPF and MAP kinase localized on spindles examined by immunoblotting and kinase assays were undetectable and less than 20%, respectively. These results indicate that the immunocytochemical technique is a powerful method for showing relative localization, but it is not suitable for quantitative analysis, and that the removal of metaphase spindles from mature oocytes does not have a severe negative impact on the subsequent MPF and MAP kinase activity and on the cell cycle progression in early embryo development.     

Effect of elevated Ca(2+) concentration in fusion/activation medium on the fusion and development of porcine fetal fibroblast nuclear transfer embryos

The present study examined the effect of elevated Ca(2+) concentration in fusion/activation medium on the fusion and development of fetal fibroblast nuclear transfer (NT) porcine embryos. Frozen-thawed and serum starved fetal fibroblasts were transferred into the perivitelline space of enucleated oocytes. Cell fusion and activation were induced simultaneously with electric pulses in 0.3 M mannitol-based medium containing 0.1 or 1.0 mM CaCl(2). Some fused embryos were further activated 1 hr after the fusion treatment by exposure to an electric pulse. The NT embryos were cultured in vitro for 6 days. Fusion and blastocyst formation rates were significantly (P<0.05) increased by increasing the Ca(2+) concentration from 0.1 mM (67.1 and 6.3%) to 1.0 mM (84.7 and 15.8%). However, no difference in the number of cells in blastocysts was observed between the two groups. A higher percentage of blastocyst was also observed when control oocytes were parthenogenetically activated in the presence of elevated Ca(2+) (19.3% vs. 32.4%, P<0.05). When the reconstituted oocytes were fused in the medium containing 1.0 mM CaCl(2), increasing the number of pulses from 2 to 3 or an additional activation treatment did not enhance the blastocyst formation rate or cell number in blastocysts. These results demonstrate that increasing the Ca(2+) concentration in the fusion/activation medium can enhance the fusion and blastocyst formation rates of fetal fibroblast NT porcine embryos without an additional activation treatment.     

Requirement for the MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation.

MAP kinase kinase (MAPKK) has been identified as a protein factor that can induce phosphorylation and activation of inactive MAP kinase in vitro. In this study, we produced an anti-Xenopus MAPKK antibody that can specifically inhibit Xenopus MAPKK activity in vitro. Microinjection of this antibody into immature oocytes prevented progesterone-induced MAP kinase activation. Moreover, progesterone-induced histone H1 kinase activation and germinal vesicle breakdown (GVBD) were inhibited in the oocytes injected previously with this antibody. Furthermore, when a bacterially expressed Mos was introduced into immature oocytes, Mos-induced MAP kinase activation and GVBD were blocked in the oocytes injected with the anti-MAPKK antibody. These results show that MAPKK is responsible for the activation of MAP kinase in vivo and that the MAPKK/MAP kinase cascade plays a pivotal role in the MPF activation during the oocyte maturation process.     

Analyses of mitogen-activated protein kinase function in the maturation of porcine oocytes

The function of mitogen-activated protein kinase (MAPK) during porcine oocyte maturation was examined by injecting oocytes with either mRNA or antisense RNA of porcine c-mos protein, an upstream kinase of MAPK. The RNAs were injected into the cytoplasm of porcine immature oocytes immediately after collection from ovaries, then the oocytes were cultured for maturation up to 48 h. The phosphorylation and activation of MAPK were observed at 6 h after injection of the c-mos mRNA injected-oocytes, whereas in control oocytes, MAPK activation was detected at 24 h of culture. The germinal vesicle breakdown (GVBD) rate at 24 h of culture was significantly higher in c-mos mRNA-injected oocytes than in control oocytes. In contrast, although injection of c-mos antisense RNA completely inhibited phosphorylation and activation of MAPK throughout the maturation period, the GVBD rate and its time course were the same in noninjected oocytes. The degree of maturation-promoting factor (MPF) activation was, however, very low in oocytes in the absence of MAPK activation. Most of those oocytes had both abnormal morphology and decondensed chromosomes at 48 h of culture. These results suggest that MAPK activation is not required for GVBD induction in porcine oocytes and that the major roles of MAPK during porcine oocyte maturation are to promote GVBD by increasing MPF activity and to arrest oocytes at the second metaphase.     

Activity of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) after parthenogenetic activation of ovine oocytes

The maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are the key regulators of both meiotic and mitotic cell cycles. Knowledge of the dynamics of these two kinases during the transition from meiosis to mitosis would be of great importance for cloning by nuclear transfer. In this study, experiments were designed to assay the changes of MPF and MAP kinase activity of in vitro matured ovine oocytes after chemical activation and culture in 0 mM or 2 mM 6-dimethylaminopurine (6-DMAP) for 12 h. Moreover, to determine the biological significance of the fluctuations of MPF, activated oocytes were fused with GV-staged partners. The biochemical results showed that the high MPF activity of MII oocytes fell to basal level precipitously within the first hour after activation, started to increase at 6-8 h, rising to 80 +/- 4% of MII after 12 h. MAPK activity decreased to a low level 4 h after activation, increased between 6-12 h, but remained below 30 +/- 3.6% of MII values. The incubation with 6-DMAP had no effect on the kinetics of MPF and MAP kinase activity. Fusion of MII oocytes to GV partners induced rapid breakdown of the GV, whereas no breakdown occurred when GV were fused with eggs in the first hours post activation. Interestingly, the high biochemical levels of MPF activity at 8-12 h after activation were not able to induce GVBD in fusion partners.     

Activation of mitogen-activated protein kinase and its activator by ras in intact cells and in a cell-free system.

Mitogen-activated protein (MAP) kinase is a serine/threonine kinase whose function is thought to be essential for the transduction of mitogenic signals. MAP kinase is activated by phosphorylation induced by a variety of extracellular stimuli, and its direct upstream activator has been identified. Using amphibian and mammalian systems, we show here that ras can activate MAP kinase and its activator. Injection of v-Ha-ras p21 into Xenopus immature oocytes activated both MAP kinase and maturation-promoting factor (MPF) activities. The activation of MAP kinase preceded that of MPF, demonstrating that ras activates MAP kinase in an MPF-independent pathway. Moreover, we found that the MAP kinase activator is also activated in ras-injected oocytes. Activation of MAP kinase and its activator occurred also when the v-Ki-ras gene was conditionally induced in rat fibroblastic 3Y1 cells. Furthermore, we observed that ras activated MAP kinase and its activator in a cell-free system prepared from Xenopus oocytes. Using an antibody against the Xenopus 45-kDa MAP kinase activator, we demonstrated that the 45-kDa activator molecule was activated by ras. These findings suggest that the MAP kinase activator/MAP kinase system may be the downstream components of ras signal transduction pathways.     

Xenopus MAP kinase activator: identification and function as a key intermediate in the phosphorylation cascade.

MAP kinase is thought to play a pivotal role not only in the growth factor-stimulated signalling pathway but also in the M phase phosphorylation cascade downstream of MPF. MAP kinase is fully active only when both tyrosine and threonine/serine residues are phosphorylated. We have now identified and purified a Xenopus MAP kinase activator from mature oocytes that is able to induce activation and phosphorylation on tyrosine and threonine/serine residues of an inactive form of Xenopus MAP kinase. The Xenopus MAP kinase activator itself is a 45 kDa phosphoprotein and is inactivated by protein phosphatase 2A treatment in vitro. Microinjection of the purified activator into immature oocytes results in immediate activation of MAP kinase. Further experiments using microinjection as well as cell free extracts have shown that Xenopus MAP kinase activator is an intermediate between MPF and MAP kinase. Thus, MAP kinase activator plays a key role in the phosphorylation cascade.