Calcineurin role in porcine oocyte activation

Calcineurin is required for oocyte exit from meiotic block in metaphase II (MII) stage in invertebrates and also in lower vertebrates. However, the role of calcineurin in mammalian oocyte activation is still unclear. The aim of this study was to determine whether calcineurin is involved in the processes regulating porcine oocyte activation. Indirect immunofluorescence demonstrated localization of both calcineurin subunits, CnA and CnB, especially in the cortex area of MII oocytes, in vitro fertilized and also parthenogenetically activated oocytes. After activation, the fluorescence intensity of the protein in the cortex area of oocytes remains unchanged; the protein calcineurin in the cytoplasm was recorded mainly around the pronuclei. Treatment of matured oocytes with calcineurin inhibitors, cyclosporin A (CsA) and hymenistatin I (HS-I), followed by activation with calcium ionophore A23187, significantly decreased the rate of activated oocytes compared to oocytes that were treated only with calcium ionophore (Ca-Io), (CsA+Ca-Io 25.0% v. Ca-Io 83.3%; HS-I+Ca-Io 32.5% v. Ca-Io 85.0%). Compared to the control, CsA treatment of matured oocytes followed by activation with Ca-Io did not affect the activity level of metaphase-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) in activated oocytes evaluated by kinase activity assay. Simultaneous staining of calcineurin and cortical granule content in matured oocytes showed that calcineurin distributed in the cortical area of the oocyte has not been colocalized with cortical granules content. On the other hand, the calcineurin inhibition before parthenogenetic activation leads to a reduction of the cortical reaction level compared to oocytes that were not treated with CsA (complete exocytosis: CsA+Ca-Io 2.6% v. Ca-Io 83.9%; sum of cortical granule brightness: CsA + Ca-Io 0.69 v. Ca-Io 0.15). Our results showed that calcineurin is involved in the process of pig oocyte activation and cortical granule exocytosis; however this regulation seems to be MPF and MAPK independent.     

A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus

Parthenogenetic activation with various combinations of the calcium ionophore A23187 and protein synthesis or phosphorylation inhibitors was investigated as a means of producing human parthenogenones with one haploid pronucleus. Unfertilised human aged oocytes exposed to 5 microM A23187 for 5 min were treated with 10 microg/ml puromycin (puromycin group, 46 oocytes) or 2 mM 6-dimethylaminopurine (DMAP group, 42 oocytes) for 5 h. Oocytes treated only with A23187 served as a control (control group, 40 oocytes). After washing the oocytes, they were incubated for up to 37 h. Evidence of activation (pronuclear formation) and cleavage was observed 18 h and 42 h after A23187 treatment, respectively. Activation rates in the puromycin and DMAP groups were significantly higher than in the control group (91% (42/46) and 77% (34/44) vs 20% (8/40), p < 0.05, respectively). In the puromycin group, 81% (34/42) of the activated oocytes showed one pronucleus with the second polar body (2ndPB), whereas none (0/34) of the activated oocytes in the DMAP group extruded the 2ndPB. The cleavage rate in the puromycin group was significantly lower than in the DMAP group (38% vs 68%, p < 0.05). The activated oocytes which had one pronucleus with the 2ndPB in the puromycin group showed a haploid set of chromosomes (10/13). In conclusion, the combination of A23187 and puromycin is effective for producing human parthenogenones with one haploid pronucleus.     

Oocyte spontaneous activation in different rat strains

Oocyte spontaneous activation (OSA) has been reported to occur during in vitro culture of ovulated rat oocytes. The objective of this study was to compare the rate of oocyte spontaneous activation and the level of maturation promoting factor (MPF) activity in oocytes from different strains. Twelve strains were selected from two commercial sources. Females were superovulated and oocytes collected 17 h after hCG injection. Denuded oocytes were cultured in M16 medium under oil at 37 degrees C and 5% CO(2) in air. The proportion of activated oocytes was determined after 6 h of in vitro culture. Data were compared by analysis of variance (ANOVA), considering each animal as an experimental unit. MPF activity was determined in oocytes from the different strains at 0, 1.5, and 3 h after oocyte collection. The log ratio of the MPF activity at 1.5 and 3 h relative to 0 hours for each animal was analyzed by ANOVA. While significant (p < 0.01) differences were observed between strains in the rate of OSA, there were no differences between strains in the level of MPF during the time points measured (p > 0.3).     

Interplay of maturation-promoting factor and mitogen-activated protein kinase inactivation during metaphase-to-interphase transition of activated bovine oocytes.

The objective of the present study was to examine the activity changes in histone H1 kinase (also known as maturation-promoting factor [MPF]) and mitogen-activated protein kinase (MAPK) and their constituent proteins in in vitro-matured bovine oocytes after in vitro fertilization (IVF) or after parthenogenetic activation induced by calcium ionophore A23187 alone or by the ionophore followed by either 6-dimethylaminopurine (6-DMAP) or cycloheximide (CHX). Inactivation of both H1 kinase and MAPK occurred after both A23187+6-DMAP treatment and IVF; inactivation of H1 kinase preceded inactivation of MAPK. However, MAPK was inactivated much earlier in 6-DMAP-treated oocytes. Further analysis of constituent cell cycle proteins of these kinases by Western blot showed that A23187 alone could not induce changes in cdc2, cdc25, or ERK2 but induced reduction of cyclin B1. IVF and A23187+CHX induced similar changes: cyclin B1 was destroyed shortly after activation followed by accumulation of cyclin B1, phosphorylation of cdc2, and dephosphorylation of ERK2 at pronuclear formation 15 h after activation. No change in cdc25 was observed at this time. In contrast, A23187+6-DMAP treatment resulted in earlier phosphorylation of cdc2 and dephosphorylation of ERK2 at 4 h after treatment when the pronucleus formed. Moreover, accumulation of both cdc25 and cyclin B1 was detected at 15 h. Microinjection of ERK2 antibody into A23187-treated oocytes resulted in pronuclear formation. In conclusion, activation of bovine oocytes with 6-DMAP led to earlier inactivation of MAPK, while CHX induced inactivation of MAPK parallel to that following sperm-induced oocyte activation. Destruction of cyclin B is responsible for inactivation of MPF, while phosphorylation of cdc2 is likely responsible for maintaining its low activity. Inactivation of MAPK is closely associated with pronuclear development regardless of the activation protocol used.     

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.     

Differential effects of protein synthesis inhibitors on porcine oocyte activation

This study was designed to evaluate the effects of cycloheximide and puromycin on activation and protein synthesis of porcine oocytes. When matured oocytes were electrostimulated, then cultured in the presence of cycloheximide (5 μ/ml) for 6 or 24 hr, 92% of oocytes were activated as indicated by pronuclear formation, vs. 2.8% for untreated oocytes, 5.3% for oocytes not electrostimulated but cultured with cycloheximide, and 60.0% for those only electrostimulated. When cultured with L-[³⁵S]methionine in the presence of cycloheximide, puromycin (100 μg/ml), or no protein synthesis inhibitor for 24 hr, oocytes had mean radiolabeled incorporation rates of 36.5, 2.21, and 32.0 fmol/4 hr/oocyte, respectively. Thus, cycloheximide had little effect on protein synthesis after 24 hr of culture. A 1D-SDS PAGE showed that oocytes cultured with puromycin or cycloheximide are not activated, while electrostimulated oocytes are activated, as characterized by the conversion of a 25-kDa polypeptide to a 22-kDa polypeptide. The radiolabeling experiment was repeated, except that oocytes were cultured for 4 or 24 hr. At 4 hr, mean incorporation rates were lower in the cycloheximide group (2.34 fmol/4 hr/oocyte), but similar in the puromycin (15.7 fmol/4 hr/oocyte) and control groups (18.9 fmol/4 hr/oocyte). At 24 hr, the puromycin group (5.73 fmol/4 hr/oocyte) had a lower rate of incorporation, while the cycloheximide (22.6 fmol/4 hr/oocyte) and control (26.0 fmol/4 hr/oocyte) groups were similar. Cycloheximide was more effective earlier during culture, while puromycin was more effective later. When combined with ES, puromycin did have a higher rate (P = 0.10) of activation (87.8%) than with electrostimulation alone (73.0%). A final experiment evaluated the development to blastocyst after transfer to a ligated oviduct. Cycloheximide treatment in conjunction with an electric pulse did not increase the rate of compact morula or blastocyst formation. In conclusion, puromycin and cycloheximide have differential effects on protein synthesis, and although cycloheximide alone will not induce activation in porcine oocytes, it is very effective in generating activated oocytes in combination with electrostimulation. © 1995 Wiley-Liss, Inc.     

Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos.

This study determines the efficiency of sequential calcium treatments (electroporation or ionomycin) combined with protein synthesis (cycloheximide) or phosphorylation inhibitors (6-dimethylaminopurine) or the specific maturation promoting factor (MPF) inhibitor, roscovitine, in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfer embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cycloheximide and cytochalasin B induced pronuclear formation and development to the blastocyst stage at a rate similar to control embryos produced by intracytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In contrast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that observed in controls. Following reconstruction by nuclear transfer, activation with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective methods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethylaminopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/roscovitine, which are applicable to parthenote or nuclear transfer embryo production.     

Changes in MPF and MAPK activities in porcine oocytes activated by different methods

The effect of different oocyte activation methods on the dynamics of M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity in porcine oocytes were examined. Three activativation methods were tested: (1) electroporation (EP); (2) electroporation combined with butyrolactone I (BL), an inhibitor of cdc2 and cdk2 kinases; (3) electroporation followed by a treatment with cycloheximide (CHX), a protein synthesis blocker. The activity of cdc2 in MII oocytes was 0.067+/-0.011pmol/oocyte/min (mean+/-S.E.M.), which by 1h decreased in every treatment group (P<0.05) and stayed at low levels until 6h post-activation, approximately the time of pronuclear formation. The initial MAPK activity (0.123+/-0.017pmol/oocyte/min) also decreased 1h after each type of activation treatment (P<0.005). However, in the electroporation only group, activity reached its lowest level at 3h; thereafter, it started to recover and at later time points, MAPK activity did not differ from that in non-treated oocytes (P>0.1). In contrast, oocytes where electroporation was followed by protein kinase or protein synthesis inhibition had low MAPK activity by the time pronuclei were to be formed. Pronuclear formation in these groups (86.3+/-3.3% for EP+BL and 87.6+/-3.7% for EP+CHX) was higher compared to that found in the EP-only oocytes (69.4+/-3.3%; P<0.05). These findings demonstrated that electroporation alone efficiently triggered the inactivation of MPF but not that of MAPK. In order to achieve low MAPK activity to allow high frequency of pronuclear formation, electroporation should be followed by a treatment that inhibits protein synthesis or specific protein kinases. The combined activation methods provided stimuli that efficiently induced both MPF and MAPK inactivation and triggered pronuclear formation with high frequencies.     

Monensin blocks the first meiotic cell division of the Xenopus oocyte: Effect at the nuclear membrane level

The carboxylic ionophore monensin inhibits the meiotic maturation of the Xenopus oocyte. When oocytes are exposed to high concentrations of monensin (10 μM), both progesterone and MPF-induced (maturation-promoting factor-induced) maturations are blocked. Lower doses of monensin (1–10 μM) do not inhibit the formation or amplification of MPF activity in the oocyte cytoplasm; however, breakdown of the nuclear envelope does not occur. These observations show that monensin, which is known to abolish intracellular proton gradients, interferes with the mechanism of the breakdown of the nuclear envelope induced by MPF.     

Nitric-oxide-dependent activation of pig oocytes: the role of the cGMP-signalling pathway

Pig oocytes matured in vitro were parthenogenetically activated (78%) after treatment with 2 mM nitric oxide-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) for 24 h. Inhibition of soluble guanylyl cyclase with the specific inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 6-anilino-5,8-quinolinequinone (LY83583) suppressed the SNAP-induced activation in a dose-dependent manner (23% of activated oocytes after treatment with 400 microM ODQ; 12% of activated oocytes after treatment with 40 microM LY83583). 8-Bromo-cyclic guanosine monophosphate (8-Br-cGMP), a phosphodiesterase-resistant analogue of cGMP, enhances the effect of suboptimal doses (0.1 or 0.5 mM) of the NO donor SNAP. DT3, a specific inhibitor of cGMP-dependent protein kinase (PKG, PKG), is also able to inhibit the activation of pig oocytes after NO donor treatment. Involvement of the cGMP-dependent signalling pathway is specific for NO-induced oocyte activation, because both the guanylyl cyclase inhibitor ODQ and the PKG inhibitor DT3 are unable to inhibit activation in oocytes treated with the calcium ionophore A23187. These data indicate that the activation of pig oocytes with an NO donor is cGMP-dependent and that PKG plays an important role in this mode of oocyte activation.     

Effective activation method with A23187 and puromycin to produce haploid parthenogenones from freshly ovulated mouse oocytes

Freshly ovulated mouse oocytes exposed to 5 mM calcium ionophore A23187 for 5 min and controls (not exposed) were cultured in TYH medium with 10 microg/ml puromycin (the puromycin group) or 2 mM 6-dimethylaminopurine (DMAP; the DMAP group) for 4 h. Among the controls, few oocytes were activated even if they were treated with DMAP or puromycin. In the oocytes exposed to A23187, in contrast, the activation rate, i.e. the rate of oocytes showing at least one pronucleus (PN) after the treatment, was 46.2% (48/104) in the DMAP group and 90.0% (118/131) in the puromycin group. Activation rate in the puromycin group was significantly higher than in the DMAP and control groups (p < 0.0001, respectively). Furthermore, 82.4% (108/131) of the activated oocytes in the puromycin group showed one PN with extrusion of the second polar body (PB). In the puromycin group, the DNA content of the PN of parthenogenones with 1PN2PB was half that of a set of metaphase II chromosomes. Chromosomal analysis was possible in 14 parthenogenones with 1PN2PB in the puromycin group. The parthenogenones possessed a normal set (n = 20) of haploid chromosomes. The combination of A23187 and puromycin proved to be an effective method of producing haploid parthenogenones.     

The M-phase-promoting factor modulates the sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate via the actin cytoskeleton

The resumption of the meiotic cycle (maturation) induced by 1-methyladenine in prophase-arrested starfish oocytes is indicated by the breakdown of the germinal vesicle and is characterized by the increased sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate (InsP3) to InsP3 starting at the animal hemisphere (where the germinal vesicle was originally located) and propagating along the animal/vegetal axis of the oocyte. This initiates Ca2+ signals around the germinal vesicle before nuclear envelope breakdown. Previous studies have suggested that the final activation of the maturation-promoting factor (MPF), a cyclin-dependent kinase, which is the major element controlling the entry of eukaryotic cells into the M phase, occurs in the nucleus. MPF is then exported to the cytoplasm where its activity is autocatalytically amplified following a similar animal/vegetal spatial pattern. We have investigated whether activated MPF was involved in the increased sensitivity of the Ca2+ response to InsP3. We have found that the development of increased sensitivity of the Ca2+ stores to InsP3 receptors together with the Ca2+ signals in the perinuclear region was blocked in oocytes treated with the specific MPF inhibitor roscovitine. That the nuclear MPF activation is indeed required for changes of the InsP3 receptors sensitivity was shown by enucleating or by dissecting oocytes into vegetal and animal hemispheres prior to the addition of 1-MA. MPF activity 50 min after 1-methyladenine addition was much lower in the enucleated oocytes and in the vegetal hemisphere, which did not contain the germinal vesicle, as compared with the animal hemisphere, which did contain it. The Ca2+ increase induced by InsP3 under these experimental conditions correlated with the changes in actin cytoskeleton induced by MPF.     

Maturation/M-phase promoting factor: a regulator of aging in porcine oocytes

Deterioration in the quality of mammalian oocytes during the metaphase-II arrest period is well known as "oocyte aging." Oocytes in which aging has occurred are called aged oocytes, and these oocytes show enhanced activation and higher fragmentation rates after parthenogenetic activation. Previously we showed that porcine aged oocytes had low maturation/M-phase promoting factor (MPF) activity, and we suggested that this low MPF activity contributed at least in part to the aging phenomena. In the present study, we examined the relationship between MPF activity and these aging phenomena by artificially regulating MPF activity in porcine metaphase-II-arrested oocytes. Since we have shown recently that aged porcine oocytes contain abundant phosphorylated inactive MPF, so-called pre-MPF, we used vanadate and caffeine, which affect the phosphorylation status of MPF, to regulate MPF activity. Incubation of 48-h-matured oocytes with vanadate for 1 h increased the phosphorylation of MPF and decreased MPF activity. The parthenogenetic activation and fragmentation rates were significantly increased compared with those of control oocytes. Conversely, treatment of 72-h-cultured aged oocytes with caffeine (last 10 h of culture) decreased the level of pre-MPF and elevated MPF activity. These oocytes revealed significantly lower parthenogenetic activation rates and a lower percentage of fragmentation than did untreated aged oocytes. These results indicate that not only the increased ability for parthenogenetic activation but also the increased fragmentation rate observed in porcine aged oocytes may be attributable in part to the gradual decrease in MPF activity during prolonged culture. Control of MPF phosphorylation with these agents may allow for some degree of manipulation of oocyte aging.     

Inhibitory effects of calcium ionophore pretreatment of porcine oocytes on polyspermic fertilization

The present study examined the inhibitory effects of various pretreatment concentrations (0-100 microM) of the calcium ionophore A23187 on polyspermic fertilization and then examined the effect of the maturation period and the time between calcium ionophore treatment and fertilization on the inhibitory effect of calcium ionophore on polyspermic fertilization. In experiment 1, a high concentration of calcium ionophore (100 microM) increased the rate of activated oocytes, but the rate of fertilization declined. On the other hand, when oocytes were treated with a low concentration of calcium ionophore (10 microM), monospermic fertilization was significantly increased (10 microM; 31.3%) (p < 0.05). In experiment 2, oocytes were cultured for various times (0, 0.5, 3, 6 h) after calcium ionophore treatment (10 microM) before fertilization. The highest rate of monospermic fertilization was detected in the oocytes cultured for 6 h after calcium ionophore treatment before fertilization. In experiments 3 and 4, we examined the effect of the maturation period (40 h or 44 h) on the rate of fertilization and blastulation of oocytes pretreated with calcium ionophore. The treatment of oocytes with calcium ionophore significantly decreased the rate of polyspermic fertilization regardless of the maturation period (44 h: with calcium ionophore 26.25% vs without 78.8%; 40 h: with calcium ionophore 37.5% vs without 77.5%); however, calcium ionophore treatment increased the rates of monospermic fertilization and blastulation of the oocytes matured for 44 h, but not those matured for 40 h. In conclusion, activation with a low concentration of calcium ionophore (10 microM) and a further 6 h of culture before fertilization improved the rate of monospermic fertilization and blastulation.     

Chemical activation of in vitro matured dromedary camel (Camelus dromedarius) oocytes: optimization of protocols

Experiments were conducted to study the efficiency of sequential treatments of ionomycine and ethanol combined with phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine) in inducing artificial activation in dromedary M-II oocytes. Cumulus oocyte complexes (COCs), collected from slaughterhouse ovaries were cultured at 38.5 degrees C in an atmosphere of 5% CO2 in air for 24-48 h. In experiment 1, the COCs were either fertilized in vitro or activated with 5 microM ionomycine for 5 min or 7% ethanol for 7 min, both followed by exposure to 6-diethylaminopurine or roscovitine for 4h. After 14-15 h of in vitro culture, the oocytes were fixed and stained with 1% aceto-orcein to evaluate their nuclear status. In experiment 2, the oocytes were activated in the same manner as in experiment 1 but were cultured for 7 days to evaluate their post-parthenogenetic development. In experiment 3, oocytes were exposed to the ionomycine for 2, 3, 4 or 5 min to evaluate the better exposure time while as in experiment 4, the oocytes matured for 28-48 h were activated to see the effect of aging on post-parthenogenetic development. Higher proportion (P<0.01) of oocytes was activated in ionomycine/6-DMAP and ionomycine/roscovitine groups when compared with ethanol/6-DMAP, ethanol/roscovitine and in vitro fertilized groups. However, there was no difference (P>0.05) in the proportion of oocytes activated with ethanol when compared with in vitro fertilized group. No significant difference was seen on the proportion of morula on day 7 of culture, however the development to blastocyst stage was higher (P<0.01) in ionomycine/6-DMAP and ionomycine/roscovitine when compared with ethanol/6-DMAP and ethanol/roscovitine treated oocytes. A higher proportion of oocytes reached blastocyst stage when they were exposed to ionomycine for 3 min but they were not significantly different from the others (P>0.05). The proportion of blastocysts obtained was higher (P<0.05) in oocytes activated after 28 h of maturation when compared with oocytes activated after 32, 36, 40, 44 and 48 h of maturation. In conclusion, a protocol for chemical activation of dromedary camel oocytes with ionomycine/6-DMAP is demonstrated and optimized in the present study for further use in the development of assisted reproductive techniques in this species.     

Effect of protein kinase C inhibitors on porcine oocyte activation

The effect of protein kinase C (PKC) inhibitors on porcine oocyte activation by calcium ionophore A23187 was studied. Calcium ionophore applied in a 50 microM concentration for 10 min induced activation in 74% of oocytes matured in vitro. When the ionophore-treated oocytes were exposed to the effect of bisindolylmaleimide I, which inhibits calcium-dependent PKC isotypes (PKC-alpha, -beta(I), -beta(II), -gamma,) and calcium-independent PKC isotypes (PKC-delta, -epsilon), the portion of activated oocytes decreased (at a concentration of 100 nM, 2% of the oocytes were activated). Go6976, the inhibitor of calcium-dependent PKC isotypes PKC-alpha, -beta(I) did not prevent the action of the oocytes treated with calcium ionophore in concentrations from 1 to 100 microM. The inhibitor of PKC-beta(I) and beta(II) isotypes, hispidin, in a concentration of 2 microM-2 mM, was not effective either. The inhibitor of PKC-delta isotype, rottlerin, suppressed activation of the oocytes by calcium ionophore (no oocyte was activated at 10 microM concentration). The PKC-delta isotype in matured porcine oocytes, studied by Western blot analysis, appeared as non-truncated PKC-delta of 77.5 kDa molecular weight, on the one hand, and as truncated PKC-delta, which was present in the form of a doublet of approximately 62.5 and 68 kDa molecular weight, on the other hand. On the basis of these results, it can be supposed that PKC participates in the regulation of processes associated with oocyte activation. Calcium-dependent PKC-alpha, -beta isotypes do not seem to play any significant role in calcium activation. The activation seems to depend on the activity of the calcium-independent PKC-delta isoform.     

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.     

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.     

The involvement of calcium in the activation of mammalian oocytes

Mouse oocytes with cumulus cells intact were parthenogenetically activated following release from the oviduct into calcium-free medium. The proportion of activated oocytes increased with post ovulatory age both for oocytes initially exposed to calcium-free and calcium-containing medium (control). Apart from oocytes released shortly after ovulation (approximately 1 h) when less than 1% of the oocytes from treated and control were activated, activation was always higher in oocytes incubated in calcium-free medium (p less than 0.001). The omission of magnesium from the medium had no effect on the activation response of oocytes obtained approximately 3 h after ovulation but its absence did increase the activation rate of oocytes of later post ovulatory age (approximately 9 h after ovulation) although it was still lower than that obtained with media devoid of calcium. When the extracellular calcium was replaced by other divalent cations (strontium, barium and manganese) high rates of activation were obtained even at post ovulatory times which produced relatively low rates of activation in calcium-free medium alone. Similar results were obtained when hamster oocytes were exposed to all the aforementioned treatments. It is concluded that calcium plays an essential role in the activation of the mammalian oocyte but the mechanism of its action remains obscure. Further development of oocytes activated by calcium-free treatment was limited and was similar to that of oocytes activated in other ways.