Polar Body Emission Research Articles

Small GTPase RhoA is required for ooplasmic segregation and spindle rotation, but not for spindle organization and chromosome separation during mouse oocyte maturation, fertilization, and early cleavage

RhoA, a small GTPase, plays versatile roles in many aspects of cell function such as stress fiber formation, cytokinesis, and cell polarization. In this study, we investigated the subcellular localization of RhoA and its possible roles during oocyte maturation and fertilization. RhoA was localized in the cytoplasm of eggs from the germinal vesicle (GV) stage to 2-cell stage, especially concentrating in the midbody of telophase spindle when oocyte extruded PB1 and PB2. The RhoA kinases (ROCKs) specific inhibitor Y-27632 blocked GV breakdown (GVBD) and first polar body extrusion, but did not affect apparatus formation and anaphase/telophase I entry. Anti-RhoA antibody microinjection into the oocytes showed similar results. RhoA inhibitor caused abnormal organization of microfilaments, failure of spindle rotation, PB2 extrusion as well as cleavage furrow formation, while sister chromatid separation was not affected. Microinjection of RhoA antibody also blocked PB2 emission. Our findings indicate that RhoA, by regulating microfilament organization, regulates several important events including GVBD, polar body emission, spindle rotation, and cleavage.

Simultaneous analysis of cytoskeletal patterns and chromosome positioning in human fertilization failures

To sequentially and reliably apply both tubulin immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH) to human fertilization failures, thus providing a tool for a multiple analysis of arrest. Analysis of human fertilization failures at several stages of arrest. Academic and clinical institutions. Consenting patients undergoing assisted reproduction technologies. Failed fertilizations displaying signs of activation without pronuclear development, or with the absence of polar body emission or cleavage 48 hours after insemination or microinjection were analyzed. Fertilization failures were fixed and processed for ICC. After data was collected the same samples were then subjected to FISH analysis using probes for chromosomes X, Y, and 18. Simultaneous ICC and FISH data on the same sample. Sequential application of straightforward standard ICC and FISH techniques was not possible, as the morphologic features had been altered, microtubular patterns were not preserved, and many samples were rendered opaque. Only chromatin at the cell surface or outside the oocyte/zygote, such as metaphase II spindles or polar body nuclei, could be routinely probed for FISH after ICC. However, an increase in detergent-induced sample permeabilization as well as the removal of several steps usually performed for FISH made it possible to directly compare microtubular patterns and chromosome position, regardless of chromatin status. Analysis of specific proteins by immunocytochemistry and of chromosome status/positioning by FISH can be carried out sequentially in human fertilization failures, irrespective of the stage of arrest.

Timing of MAP kinase inactivation effects on emission of polar body in porcine oocytes activated by Ca2+ ionophore

Artificial activation is required for successful intracytoplasmic sperm injection (ICSI) to induce haploidy pronuclear formation with extraction of second polar body. The present study showed that an additional treatment with Phorbol 12-myristate 13-acetate (PMA) followed by Ca(2+) ionophore treatment improved the rate of pronuclear formation, however, these oocytes had more than two pronuclei because of the suppression of polar body emission. The cultivation with MEK inhibitor U0126 followed by Ca(2+) ionophore also increased the rate of pronuclear formation but suppressed the emission of second polar body. These results suggested that the decrease of MAP kinase activity at early stage of artificial activation, concomitantly with decreasing p34(cdc2) kinase activity, prevented the second polar body extraction. We investigated that the timing of MAP kinase inactivation affected the extraction of the polar body and pronuclear formation rate. The addition of PMA 8 hr after Ca(2+) ionophore treatment induced the delay of MAP kinase inactivation, which resulted in haploidy pronuclear formation with emission of polar body. These results demonstrated for the first time that the delay of MAP kinase inactivation induced by PMA improved pronuclear formation with the extraction of second polar body in porcine oocytes activated by Ca(2+) ionophore. This method can be available for successfully ICSI in low response species of oocyte activation to Ca(2+) ionophore including pig.

Formation of polar‐body‐like structures induced in polyspermic starfish oocytes that had been inseminated at the germinal vesicle stage

Immature starfish oocytes, which are arrested at the first meiotic prophase and contain a large nucleus called the germinal vesicle (GV), are known to accept multiple sperm on insemination. We found that if these polyspermic starfish oocytes are induced to mature, they often form small protrusion(s) adjacent to the first polar body emitted shortly earlier. We refer to these protrusion(s) as 'polar-body-like structures (PLS).' Fluorescent staining of PLS indicated that they were not merely cytoplasmic protrusions, but contained some chromatin. Maturing process of these polyspermic oocytes was examined by immnofluorescent staining, which showed that: (i) numerous sperm asters were observed after the onset of GV breakdown; (ii) before the first polar body (PB1) emission, a complex microtubular structure resembling a multipolar spindle was formed; and (iii) several isolated asters were observed after PB1 emission. These results indicate that PLS formation may be induced by interaction of meiosis-I spindle with paternal centrosomes incorporated at GV stage.

Comparative study of cytology of cynomolgus monkey oocytes matured in vivo and in vitro

This study was conducted to compare 1) the capacity for maturation of oocytes from cynomolgus monkeys cultured in vitro before or after injection of r-hCG, 2) spindle integrity, and 3) the distribution of mitochondria in oocytes. In total, 31 stimulation cycles from 14 animals were performed. The oocytes were analyzed before or after hCG treatment and maturation in vitro. Beginning within 3 days after initiation of menstruation, adult monkeys were given 60 IU r-hFSH for 6 days, followed by 60 IU r-hFSH and 60 IU r-hLH for 2 days to stimulate multiple follicle growth. The GnRH antagonist, Antide, was administered daily (0.5 mg/kg body weight) to prevent an endogenous LH surge. Follicle development was monitored by serum estradiol concentrations and ultrasonography. Follicle aspiration was performed on anesthesized monkeys without hCG or 12, 24, or 36 h after receiving 1000 IU r-hCG. The oocytes were cultured in M199 medium containing 10% FBS and 75 μIU/ml FSH and LH for a total (in vivo and in vitro) of 40–44 h post-hCG. The maturity of oocytes was indicated by cumulus expansion and the emission of polar bodies. After removing the cumulus cells, oocytes were fixed in 3.7% formaldehyde. The spindles were visualized by using an α-tubulin antibody. Oocytes possessing a bipolar-shaped spindle with well-aligned chromosomes at the equator were considered to be normal. Finally the distribution of mitochondria was studied using MitoTracker® Red 580 and confocal microscopy. Oocytes cultured without in vivo exposure to hCG had a maturation rate of 79.6% (82/111 GV). In comparison, 82.0% (41/51 GV) and 93.8% (60/64 GVBD) of oocytes retrieved at 12h or 24 h after hCG had matured, respectively. In total, 84.5% of oocytes (82/97) retrieved at 36h after hCG had reached MII at the time of aspiration. There was no significant difference in maturation of oocytes that had been matured longer in vivo (p<0.05). The majority of oocytes in all four groups possessed abnormal spindles with asymmetric, tripolar and multipolar spindles, as well as irregular bipolar formations and unaligned chromosomes (Table 1). Spindle abnormalities were generally associated with chromosome displacement (97.9%: 142 with chromosome displacement /145 with abnormal spindles). Mitochondria were distributed mainly around the cortical region in MII oocytes, whereas in GV and GVBD oocytes they were throughout the ooplasm. The duration of culture did not significantly affect the proportion of oocytes undergoing maturation, nor did it affect the incidence of cytological anomalies. The high incidence of defects of spindle formation and chromosome alignment indicates a poor prognosis for post-fertilization development, which is comparable to human oocytes. This non-human primate appears to be a good model for studying oogenesis and embryogenesis in humans.

Low oxygen tension during in vitro maturation of porcine follicular oocytes improves parthenogenetic activation and subsequent development to the blastocyst stage

To establish a reliable in vitro maturation system for activation and subsequent development as nuclear recipients for the effective production of pig clones, we assessed maturation, activation and parthenogenetic development in response to the following: (1) type of immature oocytes (cumulus–oocyte complexes (COCs) or parietal granulosa plus cumulus–oocyte complexes (GCOCs)); (2) oxygen (O 2) tension (5 or 20%); and (3) maturation period (36–60 h). The rate of nuclear maturation to metaphase-II (M-II) in the GCOC group (73.0 ± 3.1%) was higher than that in the COC group ( P < 0.05, 60.6 ± 3.5%), but the rates did not differ between the 5 and 20% O 2 tension groups. M-II rate increased ( P < 0.05) to about 70% after 42 h and then remained constant until 60 h of culture. When oocytes were matured under 5% O 2 tension and stimulated, the rate of normal oocyte activation (a female pronucleus formation and emission of the second polar body) was higher ( P < 0.05, 38.5 ± 3.9%) than when oocytes were matured under 20% O 2 tension (24.5 ± 3.9%). On the other hand, the rate of normal activation was not significantly different between the COC and GCOC groups, and the highest ( P < 0.05) normal activation rate was obtained in oocytes cultured for 48 and 54 h (48.4 ± 5.5% and 47.9 ± 8.2%, respectively). When COC and GCOC matured for 48 h under 5 and 20% O 2 tension were stimulated and subsequently cultured in vitro for 6 days, the rate of blastocyst formation did not differ between the oocyte types nor between the O 2 tension groups. However, blastocyst quality, as measured by mean total cell number, was significantly higher in the 5% O 2 group ( P < 0.05, 34.6 ± 2.0 for COC; 33.8 ± 1.8 for GCOC) compared with the 20% O 2 group (25.9 ± 1.8 for COC; 27.0 ± 2.0 for GCOC). In conclusion, low O 2 tension (5%) during in vitro maturation of porcine oocytes promoted their ability to be activated normally and improved the quality of parthenogenetic blastocysts developed in vitro in modified NCSU-37 solutions. This knowledge may be applicable for preparation of in vitro matured oocytes with good quality as recipient oocytes for generating pig clones.

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes.

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.

Ubiquitin–proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation

Degradation of proteins mediated by ubiquitin–proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.

Regulation of ubiquitin-proteasome pathway on pig oocyte meiotic maturation and fertilization.

Degradation of proteins mediated by the ubiquitin-proteasome pathway (UPP) plays essential roles in the eukaryotic cell cycle. The main aim of the present study was to analyze the functional roles and regulatory mechanisms of the UPP in pig oocyte meiotic maturation, activation, and early embryo mitosis by drug treatment, Western blot analysis, and confocal microscopy. By using the hypoxanthine-maintained meiotic arrest model, we showed that the meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated in a dose- and time-dependent manner by two potent and cell-permeable proteasome inhibitors. Both the mitogen-activated protein kinase (MAPK) kinase inhibitor U0126 and the maturation-promoting factor inhibitor roscovitine overcame the stimulation of germinal vesicle breakdown induced by proteasome inhibitors. The phosphorylation of MAPK and p90rsk and the expression of cyclin B1 increased in a dose- and time-dependent manner when treated with proteasome inhibitors during oocyte in vitro-maturation culture. Both U0126 and roscovitine inhibited the phosphorylation of MAPK and p90rsk, and the synthesis of cyclin B1 stimulated by proteasome inhibitors. When matured oocytes were pretreated with proteasome inhibitors and then fertilized or artificially activated, the second polar body emission and the pronuclear formation were inhibited, and the dephosphorylation of MAPK and p90rsk as well as the degradation of cyclin B1 that should occur after oocyte activation were also inhibited. We also investigated, to our knowledge for the first time, the subcellular localization of 20S proteasome alpha subunits at different stages of oocyte and early embryo development. The 20S proteasome alpha subunits were accumulated in the germinal vesicle, around the condensed chromosomes at prometaphase, with spindle at metaphase I and II, the region between the separating chromosomes, and especially the midbody at anaphase I and telophase I, the pronucleus, and the nucleus in early embryonic cells. In conclusion, our results suggest that the UPP is important at multiple steps of pig oocyte meiosis, fertilization, and early embryonic mitosis and that it may play its roles by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.

Activity of MAPK/p90rsk during fertilization in mice, rats, and pigs.

AbstractMammalian oocytes enclosed in the ovary are arrested at the diplotene stage of the first meiotic prophase, which is also termed the germinal vesicle (GV) stage. Following proper stimulation (gonadotropin in mammals), the fully grown oocytes reinitiate meiosis. The resumption of meiotic maturation is characterized by germinal vesicle breakdown (GVBD), followed by chromatin condensation, microtubule organization, and emission of first polar body. Then, the oocyte meiosis arrests again at metaphase II (MII) (1), which is released by fertilization or parthenogenetic stimulation and followed by the completion of second meiotic division.KeywordsMyelin Basic ProteinZona PellucidaOkadaic AcidPolar BodyGerminal VesicleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Effects of PKC activation on the meiotic maturation, fertilization and early embryonic development of mouse oocytes.

Protein kinase C (PKC) is a family of Ser/Thr protein kinase widely distributed in eukaryotes. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. However, the mechanism of PKC's actions and the PKC isoforms responsible for these actions are poorly understood. In this study, we reveal in mouse eggs and early embryos: (1) the effects of PKC on the meiotic and mitotic cell cycle progression during oocyte maturation, egg activation and embryonic cleavages; (2) the functional importance of classical PKC subclasses in these processes; and (3) the subcellular localization of the PKC alpha isoform during development from GV stage oocytes to the blastocyst stage embryos. The results indicate that the PKC activator phorbol 12-myristate 13-acetate (PMA) inhibits the meiotic resumption of cumulus-free mouse oocytes by a mechanism dependent not only on classical PKC activity but also on other PKC isoforms. PKC activation after germinal vesicle breakdown leads to the inhibition of mitogen-activated protein kinase phosphorylation and the arrest of cell cycle at MI stage. The second polar body emission and the cleavages of early embryos are blocked after prolonged PKC activation. The subcellular localization of PKC alpha isoform in mouse oocytes and embryos is developmental-stage associated. All these results suggest that PKC has multiple functional roles in the cell cycle progression of mouse oocytes and embryos.

Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes.

To better understand the differences in cytoskeletal organization between in vivo (IVO) and in vitro (IVM) matured oocytes, we analyzed remodeling of the centrosome-microtubule complex in IVO and IVM mouse oocytes. Fluorescence imaging revealed dramatic differences in meiotic spindle assembly and organization between these two populations. Metaphase spindles at both meiosis I (M-I) and meiosis II (M-II) in IVO oocytes were compact, displayed focused spindle poles with distinct gamma-tubulin foci, and were composed of acetylated microtubules. In contrast, IVM oocytes exhibited barrel-shaped spindles with fewer acetylated microtubules and gamma-tubulin diffusely distributed throughout the spindle proper. With respect to meiotic progression, IVO oocytes were more synchronous in the rate and extent of anaphase to telophase of M-I and first polar body emission than were IVM counterparts. Furthermore, IVO oocytes showed a twofold increase in cytoplasmic microtubule organizing centers (MTOCs), and constitutive MTOC proteins (gamma-tubulin and pericentrin) were excluded from the first polar body. Inclusion of MTOC constitutive proteins in the polar body and diminished number of cytoplasmic MTOCs was observed in IVM oocytes. These findings were corroborated in IVO oocytes obtained from naturally ovulated and spontaneously cycling mice and highlight a fundamental distinction in the spatial and temporal regulation of microtubule dynamics between IVO and IVM oocytes

Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes.

Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins were translocated to the area between separating chromosomes. All these results suggest that CaMKII is a multifunctional regulator of meiotic cell cycle and spindle assembly and that it may exert its effect via regulation of MPF and MAPK/p90rsk activity during the meiotic maturation and activation of pig oocytes.

Characterization of Polo-like kinase-1 in rat oocytes and early embryos implies its functional roles in the regulation of meiotic maturation, fertilization, and cleavage.

Polo-like kinase 1 (Plk1) is a family of serine/threonine protein kinases that play important regulatory roles during mitotic cell cycle progression. In this study, Plk1 expression, subcellular localization, and possible functions during rat oocyte meiotic maturation, fertilization, and embryonic cleavages were studied by using RT-PCR, Western blot, confocal microscopy, drug-treatments, and antibody microinjection. Both the mRNA and protein of this kinase were detected in rat maturing oocytes and developing embryos. Confocal microscopy revealed that Plk1 distributed abundantly in the nucleus at the germinal vesicle (GV) stage, was associated with spindle poles during the formation of M-phase spindle, and was translocated to the spindle mid-zone at anaphase. In fertilized eggs, Plk1 was strongly stained in the cytoplasm between the apposing male and female pronuclei, from where microtubules radiated. Throughout cytokinesis, Plk1 was localized to the division plane, both during oocyte meiosis and embryonic mitosis. The specific subcellular distribution of Plk1 was distorted after disrupting the M-phase spindle, while additional aggregation dots could be induced in the cytoplasm by taxol, suggesting its intimate association with active microtubule assembly. Plk1 antibody microinjection delayed the meiotic resumption and blocked the emission of polar bodies. In conclusion, Plk1 may be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during rat oocyte meiotic maturation, fertilization, and early embryonic mitosis.

Inability of mature oocytes to create functional haploid genomes from somatic cell nuclei

Inability of mature oocytes to create functional haploid genomes from somatic cell nuclei

Cybip, a starfish cyclin B-binding protein, is involved in meiotic M-phase exit

We designed a screen to identify starfish oocyte proteins able to bind monomeric cyclin B by affinity chromatography on a cyclin B splice variant displaying low affinity for cdc2. We identified a 15 kDa protein previously described as a cdk-binding protein [Biochim. Biophys. Acta Mol. Cell Res. 1589 (2002) 219–231]. Cybip is encoded by a single polymorphic gene and the native protein is matured by cleaving a signal peptide. We firmly establish the fact that it is a true cyclin B-binding protein, since the recombinant protein binds recombinant cyclin B in absence of any cdk. Finally, we show that the microinjection of GST-cybip, and of anti-cybip antibody, in maturing starfish oocytes, inhibits H1 kinase and MPF inactivation, and first polar body emission.

Possible role for Ca2+ calmodulin-dependent protein kinase II as an effector of the fertilization Ca2+ signal in mouse oocyte activation

The present study shows that Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) is physiologically activated in fertilized mouse oocytes and is involved in the Ca(2+) response pathways that link the fertilization Ca(2+) signal to meiosis resumption and cortical granule (CG) exocytosis. After 10 min of insemination, CaM kinase II activity increased transiently, then peaked at 1 h and remained elevated 30 min later when most of the oocytes had completed the emission of the second polar body. In contrast, in ethanol-activated oocytes the early transient activation of CaM kinase II in response to a monotonic Ca(2+) rise was not followed by any subsequent increase. Inhibition of CaM kinase II by 20 micromol/l myristoylated-AIP (autocamtide-2-related inhibitory peptide) negatively affected MPF (maturing promoting factor) inactivation, cell cycle resumption and CG exocytosis in both fertilized and ethanol-activated oocytes. These results indicate that the activation of CaM kinase II in mouse oocytes is differently modulated by a monotonic or repetitive Ca(2+) rise and that it is essential for triggering regular oocyte activation.

Inhibition of MEK or cdc2 Kinase Parthenogenetically Activates Mouse Eggs and Yields the Same Phenotypes as Mos−/− Parthenogenotes

Mammalian eggs are arrested in metaphase II of meiosis until fertilization. Arrest is maintained by cytostatic factor (CSF) activity, which is dependent on the MOS–MEK–MAPK pathway. Inhibition of MEK1/2 with a specific inhibitor, U0126, parthenogenetically activated mouse eggs, producing phenotypes similar to Mos−/− parthenogenotes (premature, unequal cleavages and large polar bodies). U0126 inactivated MAPK in eggs within 1 h, in contrast to the 5 h required after fertilization, while the time course of MPF inactivation was similar in U0126-activated and fertilized eggs. We also found that inactivation of MPF by the cdc2 kinase inhibitor roscovitine induced parthenogenetic activation. Inactivation of MPF by roscovitine resulted in the subsequent inactivation of MAPK with a time course similar to that following fertilization. Notably, roscovitine also produced some Mos−/−-like phenotypes, indistinguishable from U0126 parthenogenotes. Simultaneous inhibition of both MPF and MAPK in eggs treated with roscovitine and U0126 produced a very high proportion of eggs with the more severe phenotype. These findings confirm that MEK is a required component of CSF in mammalian eggs and imply that the sequential inactivation of MPF followed by MAPK inactivation is required for normal spindle function and polar body emission.

Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes.

With improved prospects for the use of human oocyte in-vitro maturation in assisted reproductive technologies, the need to define more clearly the coordination of nuclear and cytoplasmic maturation has arisen. Immunofluorescence and confocal microscopy were used to evaluate cell cycle-dependent modifications in chromatin and microtubules in human germinal vesicle oocytes (n = 455) undergoing in-vitro maturation. Four distinct classes of germinal vesicle stage oocytes were identified based on the expression of G2/interphase characteristics, but, of these, only one class of oocytes was competent to complete meiotic progression to metaphase-II in vitro. The majority of germinal vesicle stage oocytes resumed meiosis within 6 h (88.9%) of culture and exhibited an accelerated pace of progression to metaphase-II (66.7%) over 24 h, but in general were unable to maintain meiotic arrest and defaulted into interphase within 24 h of polar body emission. Characterization of microtubule dynamics and chromatin phosphorylation demonstrates specific cell cycle deficiencies in in-vitro matured human oocytes. This work forms a basis for future studies aimed at optimizing nuclear and cytoplasmic maturation during in-vitro maturation.

Evidence that multifunctional calcium/calmodulin‐dependent protein kinase II (CaM KII) participates in the meiotic maturation of mouse oocytes

Calcium-dependent signaling pathways are thought to be involved in the regulation of mammalian oocyte meiotic maturation. However, the molecular linkages between the calcium signal and the processes driving meiotic maturation are not clearly defined. The present study was conducted to test the hypothesis that the multi-functional calcium/calmodulin-dependent protein kinase II (CaM KII) functions as one of these key linkers. Mouse oocytes were treated with a pharmacological CaM KII inhibitor, KN-93, or a peptide CaM KII inhibitor, myristoylated AIP, and assessed for the progression of meiosis. Two systems for in vitro oocyte maturation were used: (1) spontaneous gonadotropin-independent maturation and (2) follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest. FSH-induced, but not spontaneous germinal vesicle breakdown (GVB) was dose-dependently inhibited by both myristoylated AIP and KN-93, but not its inactive analog, KN-92. However, emission of the first polar body (PB1) was inhibited by myristoylated AIP and KN-93 in both oocyte maturation systems. Oocytes that failed to produce PB1 exhibited normal-appearing metaphase I chromosome congression and spindles indicating that CaM KII inhibitors blocked the metaphase I to anaphase I transition. Similar results were obtained when the oocytes were treated with a calmodulin antagonist, W-7, and matured spontaneously. These results suggest that CaM KII, and hence the calcium signaling pathway, is potentially involved in regulating the meiotic maturation of mouse oocytes. This kinase both participates in gonadotropin-induced resumption of meiosis, as well as promoting the metaphase I to anaphase I transition. Further evidence is therefore, provided of the critical role of calcium-dependent pathways in mammalian oocyte maturation.