Protein synthesis requirement for maturation promoting factor (MPF) initiation of meiotic maturation in Rana oocytes.

Mechanisms controlling disintegration or breakdown of the germinal vesicle (GVBD) in Rana oocytes were investigated. A secondary cytoplasmic maturation promoting factor (MPF), produced in response to steroid stimulation, was shown to induce maturation when injected into immature recipient oocytes. Exposure of immature Rana oocytes to cycloheximide following injection of MPF or steroid treatment completely inhibited such maturation. Results indicate that injected MPF required protein synthesis for germinal vesicle breakdown and thus acted at some translational level. These results contrast with data obtained in Xenopus oocytes where injected MPF induced maturation in the presence of cycloheximide. Cytoplasmic MPF was also produced in Rana oocytes following treatment with lanthanum salts. This activity was similarly inhibited by cycloheximide. Time course studies conducted to compare the onset of cycloheximide insensitivity in steroid-treated and MPF-injected oocytes demonstrated that MPF-injected oocytes become insensitive to cycloheximide prior to steroid-treated germ cells. These results suggest that MPF acts as an intermediary in progesterone-induced maturation. Insensitivity to cycloheximide occurred several hours prior to the onset of germinal vesicle breakdown in both MPF-injected and steroid-treated oocytes. The data indicate that injected MPF in Rana does not induce nuclear disintegration directly, but rather requires amplification and/or autocatalytic synthesis of additional MPF or other factors for maturation to be induced. Molecular mechanisms involved in nuclear disintegration are discussed in relation to these species differences.     

M-phase promoting factor (MPF) and mitogen activated protein kinases (MAPK) activities of domestic cat oocytes matured in vitro and in vivo

This work was undertaken in order to examine M-phase promoting factor (MPF) and mitogen-activated protein kinases (MAPK) activities during meiotic progression of cat oocytes cultured in two different media for two different incubation times and preovulatory cat oocytes that reached MII in vivo. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 or SOF for 24 h and 40 h. In vivo matured oocytes were recovered by follicular aspiration from ovaries of domestic cats ovariectomized 24 h to 26 h after hormonal treatment. Results showed that the kinetic of MPF and MAPK activity was similar during meiotic progression of cat oocytes matured in TCM 199 and SOF. After 24 h of incubation, MII oocytes had significantly (p < 0.001) higher MPF and MAPK levels than MII oocytes cultured for 40 h in both culture media. MPF and MAPK activity was significantly (p < 0.01) lower in the oocytes matured in vitro than in those matured in vivo. This study provides evidence that the two different maturation media did not determine differences in MPF and MAPK fluctuations and levels during meiotic progression of cat oocytes and that the time of maturation influenced the level of the two kinases. Moreover, it shows that MPF and MPK activity is higher in in vivo matured oocytes than in in vitro matured oocytes, suggesting a possible incomplete cytoplasmic maturation after culture.     

Maturation/M-phase promoting factor regulates aging of porcine oocytes matured in vitro

Control of oocyte aging during manipulation of matured oocytes should have advantages for recently developed reproductive technologies, such as cloning after nuclear transfer. We have shown that the enhanced activation ability and fragmentation of porcine in vitro matured and aged oocytes bore a close relationship to the gradual decrease in maturation/M-phase promoting factor (MPF) activity and that porcine aged oocytes contained plenty of MPF, but it was in an inactive form, pre-MPF, as a result of phosphorylation of its catalytic subunit p34(cdc2) and, therefore, had low MPF activity. We incubated porcine oocytes with vanadate and caffeine, which affected the phosphorylation status and MPF activity, and evaluated their activation abilities and fragmentation frequencies. Incubation of nonaged oocytes with vanadate increased p34(cdc2) phosphorylation and reduced MPF activity to levels similar to those of aged oocytes and increased their parthenogenetic activation and fragmentation rates compared with those of the control oocytes. Conversely, treating aged oocytes with caffeine reduced p34(cdc2) phosphorylation and increased MPF activity. These oocytes showed significantly lower parthenogenetic activation and fragmentation rates than aged mature oocytes. These results suggest that MPF activity is a key mechanism of oocyte aging and controlling MPF activity by altering p34(cdc2) phosphorylation with these chemicals may enable oocyte aging to be manipulated in vitro. We expect those ideas will be applied practically to pig cloning.     

High developmental competence of pig oocytes after meiotic inhibition with a specific M-phase promoting factor kinase inhibitor,butyrolactone I

Butyrolactone I specifically inhibits M-phase promoting factor activation and prevents the resumption of meiosis. These experiments were conducted to examine effects of butyrolactone I on pig oocytes in a serum-free maturation system. The first experiment was conducted to determine the effect of butyrolactone I (0-100 microM) on nuclear maturation. At concentrations of > or =12.5 microM, germinal vesicle breakdown was prevented in >90% of the oocytes after 24 h of culture. In the second experiment, the kinetics of in vitro maturation of butyrolactone I-treated oocytes was investigated. Oocytes were treated with 0 or 12.5 microM butyrolactone I and FSH for 20 h and then cultured with LH in the absence of butyrolactone I for another 24 h. Fewer butyrolactone I-treated oocytes reached MII stage at 36 h compared with controls (5.8% vs. 62.4%, P < 0.01). However, by 44 h, 83.4% of butyrolactone I-treated oocytes reached MII compared with 88.6% of controls. In the third experiment, butyrolactone I-treated oocytes were fertilized and cultured in vitro. No differences (P > 0.05) were found between controls and treated groups in cleavage rate, blastocyst rate, or mean number of cells per blastocyst. Effects of butyrolactone I on mitogen-activated protein kinase activation and localization of microfilaments and active mitochondria were examined by Western blot analysis and laser scanning confocal microscopy, respectively. The results suggested that although butyrolactone I reversibly inhibited germinal vesicle breakdown and mitogen-activated protein kinase activation, it did not affect mitochondrial and microfilament dynamics. Butyrolactone I is a potent inhibitor of nuclear maturation of porcine oocytes, and the inhibition is fully reversible.     

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.     

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.     

Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes

In cows, protein synthesis is required for germinal vesicle breakdown (GVBD). This study examines more closely the need for protein synthesis and the nuclear changes in the bovine oocyte during 24 h of culture. Bovine oocytes with compact and complete cumulus were washed and incubated in groups of 10 for up to 24 h in 50-microliters drops of TCM-199 supplemented with follicle-stimulating hormone (NIAMADD, 0.5 micrograms/ml), luteinizing hormone (LH) NIAMADD, 5 micrograms/ml), estradiol-17 beta (1 microgram/ml), pyruvate (20 microM), and 10% heat-treated fetal calf serum. Medium was overlaid with paraffin oil. Oocytes (n = 891) were fixed at the end of each 3-h interval from 0 to 24 h of culture, or at 24 h after addition of cycloheximide (10 micrograms/ml at 10 different times during maturation (0, 1, 2, 3, 6, 9, 12, 15, 18, 21 h; n = 175). At each time point, the chromosomal status of oocytes was evaluated, frequencies were computed, and the time spent on each step was determined. The germinal vesicle (GV) was present from 0 to 6.6 h, GVBD at 6.6 to 8.0 h, chromatin condensation at 8.0 to 10.3 h, metaphase I at 10.3 to 15.4 h, anaphase I at 15.4 to 16.6, telophase I at 16.6 to 18.0 h, and metaphase II at 18.0 to 24 h. Cycloheximide blocked oocyte maturation at GVBD, if added from 0 to 3 h; at chromatin condensation, if present from 6 to 24 h; and at metaphase I, when present from 9 to 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

原癌基因c-erbB2和c-myb经丝裂原活化蛋白激酶和成熟促进因子途径调节卵母细胞的成熟

本研究探讨了原癌基因c-erbB:和c-myb对小鼠卵母细胞成熟的影响及其在调控卵母细胞成熟中与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和成熟促进因子(mamration promoting factor,MPF)的上下游关系.c-erbB2反义寡脱氧核苷酸(antisense oligodeoxynucleotide,ASODN)和c.myb ASODN均呈剂量依赖方式抑制卵母细胞的生发泡破裂(germinalvesicle breakdown,GVBD)率和第一极体(first polar body,PBl)排放率,并显著延迟其成熟时间.小鼠卵母细胞显微注射重组人c-erbB2蛋白和c-myb蛋白后,培养6 h其GVBD率分别比对照组上升了23.1%(P<0.05)和32.2%(P<0.05),.培养12 h其PBl排放率分别比对照组上升了17.3%(P<0.05)和23.5%(P<0.05).RT-PCR结果显示,小鼠卵母细胞中存在c-erbB2mRNA和c-myb mRNA表达;c-erbB2ASODN能明显抑制卵母细胞中c-erbB2mRNA和c-myb mRNA的表达,c-myb ASODN能明显抑制卵母细胞中c-myb mRNA的表达,对c-erbB2 mRNA无明显影响;MAPK抑制剂PD98059以及MPF抑制剂roscovitine在抑制卵母细胞成熟的同时,均能阻断显微注射重组人c-erbB:蛋白和重组人c-myb蛋白对卵母细胞成熟的促进作用,但对卵母细胞中c-erbB2mRNA和c-myb mRNA表达无明显影响.Western blot结果显示,c-erbB2ASODN、c-mybASODN、PD98059、roscovitine均使卵母细胞中MAPK磷酸化水平和cyclinB 1含量下降.结果提示,原癌基因c-erbB2、c-myb在卵母细胞成熟中起重要作用,可能是调控卵母细胞成熟中关键蛋白激酶如MAPK、MPF的上游激活物.     

Method for the preparation of active maturation promoting factor (MPF) from in vitro matured oocytes of Xenopus laevis.

A method for the large scale extraction of Maturation Promoting Factor (MPF) from in vitro matured oocytes of Xenopus laevis is described. MPF has been previously described only as a component(s) of hormone-matured cytoplasm within amphibian oocytes (or eggs) which is able to induce the reinitiation of the meiotic process from late diplotene stage until second metaphase arrest, when microinjected into diplotene arrested (fully grown) recipient oocytes. Standard biochemical methods for the extraction and purification of this factor(s) haven been unsuccessful due to its extreme instability and sensitivity to dilution. The procedure is dependent upon the inclusion of sodium fluoride (NaF) in the extraction medium with its effect presumably due to its ability to inhibit phosphorprotein phosphatases. The successful preservation of MPF activity described in this report permits further attempts to be made to isolate and characterize this, to date, elusive cytoplasmic factor, which plays a key role in the complex cellular processes involved in the hormone-dependent differentiation of an oocyte into an egg.     

Lithium inhibits amplification or action of the maturation-promoting factor (MPF) in meiotic maturation of starfish oocytes

Microinjection of LiCl reversibly inhibits hormone-induced meiotic maturation of starfish oocytes. Microinjection of NaCl (even in ouabain-treated oocytes) or KCl, or external application of LiCl have no such effect. Blockade of meiotic maturation by Li+ occurs even when microinjection is performed after the hormone dependent period has ended, that is the period during which the hormone must be present in the medium in order that meiosis can take place. Li+ microinjection prevents oocytes from meiosis reinitiation following transfer of cytoplasm taken from maturing oocytes, which contain a maturation-promoting factor (MPF). Cytoplasm taken from Li+-injected and hormone-treated oocytes does not trigger meiosis reinitiation when transferred in control immature oocytes. Intracellular pH does not change following LiCl microinjection. Simultaneous microinjection of either K+, Na+, or EGTA does not prevent Li+-dependent inhibition in oocytes.     

Stabilization of the maturation promoting factor (MPF) from Xenopus laevis oocytes. Protection against calcium ions

Maturation promoting factor (MPF) activity was recovered from progesterone-matured Xenopus oocytes cytosol, fractionated by polyethylene glycol-20,000 or ethanolic precipitation. An improved stabilization of the biological activity was obtained by adding adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) (50 microM) to the preparation buffers. Neither Ca2+ ions nor calmodulin inhibit the partially purified MPF.     

Effects of enucleation and caffeine on maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities in ovine oocytes used as recipient cytoplasts for nuclear transfer

In general, oocytes arrested at metaphase of the second meiotic division (MII) are used as recipient cytoplasts for nuclear transfer (NT) procedures. MII oocytes contain high levels of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), which cause nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) in the transferred nucleus and have been implicated in nuclear reprogramming. However, the occurrence of NEBD and the extent of PCC are variable between individual oocytes and species and are dependent on donor cell type and cell cycle stage. Enucleation, which removes oocyte cytoplasm, may reduce MPF and MAPK activities and reduce reprogramming; conversely, increasing kinase activities may increase reprogramming. We compared the effects of enucleation of ovine oocytes at anaphase/telophase of the first meiotic division (AI-TI) and at MII. MPF and MAPK activities were maximal at MII; blind enucleation at AI-TI was more efficient than at MII and removed a smaller volume of cytoplasm. Neither protocol significantly affected the activity of either kinase and the fate of the donor nucleus; however, enucleation per se significantly reduced the occurrence of NEBD in NT embryos. Treatment with 10 mM caffeine significantly increased the activities of both kinases and the occurrence of NEBD but did not affect the frequency of development to the blastocyst stage; however, a significant increase in total cell numbers was observed. The results show that caffeine can increase MPF and MAPK activities in ovine oocytes and that this may contribute to an increased reprogramming in NT embryos.     

Meiotic maturation of mouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression.

In vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence of several drugs. The individual steps of nuclear progression, including dissolution of the nuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact bivalents, formation of the first metaphase spindle, and extrusion of the first polar body, are each susceptible to one or more of these drugs. Germinal vesicle breakdown, the initial morphological feature characteristic of meiotic maturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyryl cyclic AMP, the nuclear membrane becomes extremely convoluted and condensation of chromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle breakdown and chromatin condensation take place in an apparently normal manner in the presence of puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circular bivalent stage when oocytes are cultured continuously in the presence of puromycin or Colcemid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meiotic metaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibited by all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversible to varying degrees dependent upon the duration of exposure to the drug and upon the nature of the drug. These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensation of chromatin are not dependent upon concomitant protein synthesis or upon microtubules. On the other hand, the complete condensation of chromatin into compact bivalents apparently requires breakdown of the germinal vesicle. Failure of homologous chromosomes to separate after normal alignment on the meiotic spindle in the presence of cytochalasin B suggest that microfilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis, in the form of polar body formation, is blocked when any one of the earlier events of maturation fails to take place.     

Reprogramming of bovine somatic cell nuclei is not directly regulated by maturation promoting factor or mitogen-activated protein kinase activity

Cloned mammals with normal fertility have been produced by nuclear transfer. Thus, oocyte cytoplasm has the ability to convert differentiated somatic cell nuclei into a state that resembles the conditions that occur at fertilization (nuclear reprogramming). Despite the long-held assumption that reprogramming factors are present in mammalian oocytes, the molecular nature of these factors is not known. The present study demonstrates that the process of nuclear reprogramming is not directly regulated by maturation promoting factor or mitogen-activated protein kinase activity. The potential for nuclear-transferred oocytes to develop to the blastocyst stage was not different when somatic cells at the M phase were fused with oocytes activated with ionomycin and cycloheximide 1-5 h before (12%-22%) but was significantly decreased when oocytes were activated 6 h before (1%). Further molecular studies on the differences between oocytes with and without reprogramming potential are required and will be useful for the identification of reprogramming factors.     

Activation of mitogen-activated protein (MAP) kinase by a MAP kinase-kinase.

Previously it has been shown that acute 12-O-tetradecanoylphorbol-13-acetate treatment of intact U937 cells results in activation of mitogen-activated protein (MAP) kinase and a MAP kinase activator. MAP kinase activator induces phosphorylation of MAP kinase on tyrosine and threonine residues, thereby activating MAP kinase. Here, experiments with the irreversible kinase inhibitor, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), show that MAP kinase activator is in fact a MAP kinase-kinase. Treatment of MAP kinase activator with FSBA results in complete inactivation. This inactivation is prevented by a 10-fold excess of ATP. Inactivation of MAP kinase by FSBA does not affect the extent of threonine/tyrosine phosphorylation induced by MAP kinase-kinase.     

Dynamin is required for the activation of mitogen-activated protein (MAP) kinase by MAP kinase kinase

Internalization of activated receptors from the plasma membrane has been implicated in the activation of mitogen-activated protein (MAP) kinase. However, the mechanism whereby membrane trafficking may regulate mitogenic signaling remains unclear. Here we report that dominant-negative dynamin (K44A), an inhibitor of endocytic vesicle formation, abrogates MAP kinase activation in response to epidermal growth factor, lysophosphatidic acid, and protein kinase C-activating phorbol ester. In contrast, dynamin-K44A does not affect the activation of Ras, Raf, and MAP kinase kinase (MEK) by either agonist. Through immunofluorescence and subcellular fractionation studies, we find that activated MEK is present both at the plasma membrane and in intracellular vesicles but not in the cytosol. Our findings suggest that dynamin-regulated endocytosis of activated MEK, rather than activated receptors, is a critical event in the MAP kinase activation cascade.     

Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity

Compared with oocytes matured in vivo, in vitro-matured oocytes are compromised in their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3- (oocyte) and isoenzyme 4- (granulosa cell) specific PDE inhibitors on the kinetics of in vitro maturation and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 microM). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9 h, but they completely blocked meiotic resumption during this period (P < 0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% at germinal vesicle stage after 48 h) was overridden by 24 h when treated with FSH, but not with hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 PDE inhibitor significantly (P < 0.05) increased embryo development to the blastocyst stage by 33%-39% (to an average of 52% blastocysts) compared with control oocytes (38%) after insemination at 28 h, and significantly (P < 0.05) increased blastocyst cell numbers when inseminated at 24 h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the CCs and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential.     

Insulin-transferrin-selenium (ITS) improves maturation of porcine oocytes in vitro

The objective of this study was to determine if insulin-transferrin-selenium (ITS) promoted a nuclear and cytoplasmic maturation of porcine oocytes that better supports subsequent embryonic development. The rate of oocyte in vitro maturation (IVM) in an experimental group treated with hormones for 42 h was significantly increased compared with that in a control group without hormone treatment (47.8% vs. 11.7%, respectively, p < 0.05). Following reduction of the hormone treatment period from 42 h to 21 h, which included both the first 21 h period of hormones treatment (45.4%) and the second 21 h period of hormone treatment (44.8%), the rate of oocyte IVM was still higher than that of the control group (p < 0.05). To improve porcine oocyte nuclear maturation, 1% ITS was added to medium supplemented with hormones. The rate of nuclear maturation in the ITS-treated group was significantly higher than in the ITS-untreated group (78.6% vs. 54.4%, respectively, p < 0.05). ITS treatment also significantly reduced the per cent of oocytes with type I and type III cortical granule (CG) distribution, respectively, and significantly increased the per cent of oocytes with type II CG distribution (85.3%). These observations indicated that the synchronization rates of nuclear and ooplasmic maturation reached 67.04% (78.56 × 85.33%). In conclusion, the combination of modified Tissue Culture Medium-199 (mM199) + 10 ng/ml epidermal growth factor (EGF) + 10 IU/ml pregnant mare serum gonadotrophin (PMSG) + 10 IU/ml human chorion gonadotrophin (hCG) + 2.5 IU/ml follicle stimulating hormone (FSH) + 1% ITS is suitable for culturing porcine oocytes in vitro, and effectively enhances porcine oocyte nuclear and cytoplasmic maturation.