The effect of PD98059 on MAPK regulation in cumulus-enclosed and cumulus-free mouse oocytes

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Jaroslav Kalous ◽  
Michal Kubelka ◽  
Jan Motlík
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Experimental Conditions ◽  
Germinal Vesicle Breakdown ◽  
Mapk Activation ◽  
Mapk Phosphorylation ◽  
Kinase Activation ◽  
Mouse Oocytes ◽  
Significant Difference ◽  
Epidermal Growth

The effect of the p42/44 mitogen-activated kinase (MAPK) inhibitor, PD98059, on MAPK activation and meiosis resumption in mouse oocytes was studied. When germinal vesicle (GV)-stage denuded oocytes (DOs) were cultured continuously in 50 μM PD98059, germinal vesicle breakdown (GVBD) was postponed for 2-3 h. MAPK phosphorylation and activation was delayed as well. However, PD98059 did not impair histone H1 kinase activation. After 14 h of culture there was no significant difference in the rate of DOs reaching metaphase II (MII) arrest in either control or experimental conditions. The effect of PD98059 on MAPK inhibition was further tested in epidermal growth factor (EGF)-treated oocyte–cumulus complexes (OCCs). Exposure of GV-stage OCCs for 5 min to EGF (10 ng/ml) induced a considerable increase in MAPK phosphorylation. After OCCs were further cultured in 50 μM PD98059 a rapid dephosphorylation of MAPK was induced. Already after 1 min of treatment the non-phosphorylated form of MAPK dominated, indicating the high effectivity of PD98059. This result indicates that short EGF/PD98059 treatment of OCCs induced MAPK phosphorylation/dephosphorylation in cumulus cells only. As only a transient delay in MAPK phosphorylation and activation was observed in PD98059-treated DOs we conclude that there is also another PD98059-nonsensitive pathway(s) leading to MAPK activation in mouse oocytes. The data obtained suggest that meiosis resumption in mouse oocytes is somehow influenced by the MEK/MAPK activation pathway.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic induction in isolated mouse oocytes

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 71-82 ◽  
Author(s):  
Stephen M. Downs
Keyword(s):  
Induction Period ◽  
Gap Junction ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Paracrine Factor ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
Junctional Communication ◽  
Positive Factor

This study was carried out to compare the possible role of a secreted paracrine factor versus that of a gap-junction-transmitted signal in mediating meiotic induction in isolated mouse oocytes from PMSG-primed, immature mice. In the first set of experiments, oocyte-cumulus cell complexes (OCC) were pretreated for 3 h with 2 mM dbcAMP or FSH, washed, and the oocytes then cultured for 17-18 h in 40 μl drops containing either 300 μM dbcAMP or 4 mM hypoxanthine (HX). Each set of pretreated oocytes was cultured under three different conditions: (1) intact cumulus-cell-enclosed oocytes (CEO); (2) denuded oocytes (DO), cultured alone after removal of cumulus cells; and (3) co-cultured cumulus cells and oocytes (CC/DO), where the cumulus cells were removed in the same drop with a mouth-operated pipette and cultured alongside the oocytes. When pretreated with high dbcAMP or FSH, maturation was stimulated in CEO when cultured in either inhibitor (by 41.4-53.7%). Pretreatment failed to affect the maturation rate in DO. DO maturation was not altered appreciably by co-cultured cumulus cells when arrest was maintained with dbcAMP. However, an increase in maturation of 21-23% was observed in CC/DO in the HX-containing cultures that was not dependent on prior treatment with a meiosis-inducing stimulus. When DO were co-cultured with intact, FSH-treated OCC, there was no evidence of a positive factor secreted by the stimulated complexes, despite the fact that oocytes within the OCC were induced to resume maturation. In a second series of experiments the gap junction inhibitor, 18α-glycyrrhetinic acid (GA), was utilised. An initial experiment determined that GA dose-dependently blocked OCC metabolic coupling (0.2% coupling at 10 μM compared with 13.6% in controls). When HX-arrested CEO and DO were cultured for 17-18 h in medium containing increasing concentrations of GA, meiotic maturation was induced in CEO but not DO, suggesting that the cumulus cells provided a positive stimulus in the absence of functional gap junctional communication. No effect of GA was seen in dbcAMP-arrested oocytes. A kinetics experiment showed that when CEO were cultured in dbcAMP±FSH, meiotic induction was initiated after 3 h and germinal vesicle breakdown reached 60% by 6 h. When GA was added to the cultures at different times after the initiation of culture (0, 2, 3, 4 and 5 h), meiotic induction was immediately blocked. In addition, measurement of OCC coupling revealed that no reduction in coupling occurred during this induction period in the absence of GA. It is concluded that cumulus cells can secrete a positive factor, but that this is normally overridden by inhibitory influences transmitted through the gap junction pathway in intact complexes. Furthermore, upon exposure of complexes to a meiosis-inducing stimulus, a positive gap-junction-mediated signal now predominates to trigger germinal vesicle breakdown, and this signal is utilised throughout the induction period.

Mitogen-activated protein kinase phosphorylation patterns in pig oocytes and cumulus cells during gonadotrophin-induced resumption of meiosis in vitro

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 139-147 ◽  
Author(s):  
S. Ebeling ◽  
C. Schuon ◽  
B. Meinecke
Keyword(s):  
Protein Kinase ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Mek Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Germinal Vesicle Breakdown ◽  
Mapk Phosphorylation ◽  
Rapid Action ◽  
Mitogen Activated Protein

SummaryThe present study investigated the phosphorylation pattern of mitogen-activated protein kinase (MAPK) in cumulus–oocyte complexes (COCs) during spontaneous and FSH/LH-induced in vitro maturation (IVM). Both isoforms of MAPK were unphosphorylated in oocytes recovered immediately after liberation from follicles and became phosphorylated following 25 h incubation, corresponding to the time of germinal vesicle breakdown (GVBD). In contrast, MAPK was already phosphorylated in minimal amounts in cumulus cells at the time of liberation from follicles and phosphorylation of MAPK increased after 0.5 h incubation. Supplementation of medium with gonadotrophins intensified phosphorylation at 0.5 h incubation, demonstrating the early and rapid action of FSH/LH on MAPK phosphorylation. Phosphorylation of MAPK in cumulus cells peaked after 21 h of incubation, whereas MAPK was almost completely dephosphorylated at the end of incubation (45 h). During subsequent incubation in the absence of added gonadotrophins, between 5 and 10 h exposure to FSH/LH-supplemented medium was required to induce resumption of meiosis in COCs. Phosphorylation of MAPK in oocytes was prevented by the MEK inhibitor U0126, but the inhibitor reduced phosphorylation of MAPK in cumulus cells only during the first 2 h of IVM. The data support the hypothesis that two different MAPK phosphorylation events occurred following gonadotrophin stimulation, one in cumulus cells and the other in oocytes. In cumulus cells, FSH/LH induced early and rapid U0126-insensitive phosphorylation of MAPK, whereas U0126-susceptible MAPK phosphorylation took place in the oocyte itself around the time of GVBD.

Pleiotropic effect of okadaic acid on maturing mouse oocytes

Development ◽  
1991 ◽  
Vol 112 (4) ◽  
pp. 971-980 ◽  
Author(s):  
H. Alexandre ◽  
A. Van Cauwenberge ◽  
Y. Tsukitani ◽  
J. Mulnard
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Chromatin Condensation ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Inhibitory Effect ◽  
Negative Control ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.

Ha-rasVal-12,Thr-59 activates S6 kinase and p34cdc2 kinase in Xenopus oocytes: evidence for c-mosxe-dependent and -independent pathways

1990 ◽  
Vol 10 (1) ◽  
pp. 310-315
Author(s):  
C B Barrett ◽  
R M Schroetke ◽  
F A Van der Hoorn ◽  
S K Nordeen ◽  
J L Maller
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Antisense Oligonucleotides ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Protein Product ◽  
Germinal Vesicle Breakdown ◽  
S6 Kinase ◽  
Kinase Activation ◽  
Histone Kinase

Treatment with insulin or progesterone or microinjection of the transforming protein product of Ha-rasVal-12,Thr-59 (p21) is known to induce germinal vesicle breakdown in Xenopus oocytes. We have investigated the effect of p21 on S6 kinase and the H1 histone kinase of maturation-promoting factor in the presence and absence of antisense oligonucleotides against the c-mosxe proto-oncogene. Injection of p21 led to a rapid increase in S6 phosphorylation, with kinetics similar to those previously observed with insulin. Microinjection of c-mosxe antisense oligonucleotides inhibited germinal vesicle breakdown induced by p21 and totally abolished S6 kinase activation by insulin or progesterone but only partially inhibited activation by p21. However, the activation of p34cdc2 protein kinase by all three stimuli was blocked by antisense oligonucleotides. The results suggest that in oocyte maturation c-mosxe functions downstream of p21 but upstream of p34cdc2 and S6 kinase activation, although not all p21-induced events require c-mosxe.

Mice homozygous for an insertional mutation in the Zp3 gene lack a zona pellucida and are infertile

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2903-2910 ◽  
Author(s):  
T. Rankin ◽  
M. Familari ◽  
E. Lee ◽  
A. Ginsberg ◽  
N. Dwyer ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Genetic Defect ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Preimplantation Embryos ◽  
Germinal Vesicle Breakdown ◽  
Female Mice ◽  
Insertional Mutation ◽  
Homozygous Mutant

Mammalian oocytes synthesize and secrete a zona pellucida that surrounds the growing oocytes, ovulated eggs and preimplantation embryos. The extracellular zona matrix is composed of three glycoproteins (ZP1, ZP2, ZP3) that are involved in folliculogenesis, species-specific fertilization, and passage of the early embryo down the oviduct. We have established a mouse line in which Zp3 has been inactivated by homologous recombination with an insertional mutation. Neither Zp3 transcripts nor ZP3 protein was detected in female mice homozygous for the mutation (Zp3−/−), whereas both ZP1 and ZP2 were present in mutant oocytes. Homozygous mutant Zp3−/− mice had follicles with germinal-vesicle-intact oocytes but that lacked a zona pellucida matrix and had a disorganized corona radiata. Although mutant oocytes underwent germinal vesicle breakdown (GVBD) prior to ovulation, the cumulus-oocyte complex was markedly disrupted and the oocytes were often separate from the cumulus cells. After hormone-induced ovulation, cumulus masses were present in the oviducts of homozygous mutant mice, but zona-free eggs were observed in only half of the females and, in these, less than 10% of the normal number [correction of mumber] of eggs were detected. No zona-free 2-cell embryos were recovered from homozygous mutant Zp3−/− female mice after mating with males proven to be fertile, and none became visibly pregnant or produced offspring. These results demonstrate that a genetic defect in a zona pellucida gene causes infertility and, given the conserved nature of the zona pellucida, a similar phenotype is expected in other mammals.

The Role of Tissue Transglutaminase in the Germinal Vesicle Breakdown of Mouse Oocytes

2001 ◽  
Vol 286 (2) ◽  
pp. 229-234 ◽  
Author(s):  
Sung Woo Kim ◽  
Zee-Won Lee ◽  
ChangKyu Lee ◽  
Kyung Soon Im ◽  
Kwon-Soo Ha
Keyword(s):  
Tissue Transglutaminase ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes

IVM of mouse fully grown germinal vesicle oocytes upon a feeder layer of selected cumulus cells enhances their developmental competence

2019 ◽  
Vol 31 (6) ◽  
pp. 1068
Author(s):  
Federica Cavalera ◽  
Milena Simovic ◽  
Mario Zanoni ◽  
Valeria Merico ◽  
Silvia Garagna ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Development Rate ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Oocyte Developmental Competence ◽  
Mouse Oocytes ◽  
Feeder Layers ◽  
Morula Stage ◽  
Bidirectional Exchange

In the ovary, acquisition of oocyte developmental competence depends on a bidirectional exchange between the gamete and its companion cumulus cells (CCs). In this study we investigated the contribution of CCs surrounding oocytes of known developmental competence or incompetence to the acquisition of oocyte developmental competence. To this end, feeder layers of CCs (FL-CCs) were prepared using CCs isolated either from: (1) developmentally competent mouse oocytes whose nucleolus was surrounded by a chromatin ring (FL-SN-CCs); or (2) developmentally incompetent mouse oocytes whose nucleolus was not surrounded by a chromatin ring (FL-NSN-CCs). Denuded, fully grown oocytes (DOs) were matured to the MII stage on either FL-SN-CCs or FL-NSN-CCs, inseminated with spermatozoa and cultured throughout preimplantation development. FL-SN-CCs significantly improved the acquisition of oocyte developmental competence, with a blastocyst development rate equal to that for maturation of intact cumulus–oocyte–complexes. In contrast, DOs matured on FL-NSN-CCs or in the absence of CCs exhibited developmental failure, with embryos arresting at either the 4-cell or morula stage. These results set a culture platform to further improve the protocols for the maturation of DOs and to unravel the molecules involved in the cross-talk between the gamete and its companion CCs during the germinal vesicle to MII transition.

scholarly journals Obox4-silencing-activated STAT3 and MPF/MAPK signaling accelerate nuclear membrane breakdown in mouse oocytes

Reproduction ◽  
2016 ◽  
Vol 151 (4) ◽  
pp. 369-378 ◽  
Author(s):  
Hyun-Seo Lee ◽  
Kyeoung-Hwa Kim ◽  
Eun-Young Kim ◽  
Su-Yeon Lee ◽  
Jung-Jae Ko ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Molecular Mechanisms ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Mapk Signaling ◽  
Interferon Α ◽  
Membrane Breakdown ◽  
Nuclear Maturation ◽  
Mouse Oocytes

Mouse oocytes begin to maturein vitroonce liberated from ovarian follicles. Previously, we showed that oocyte-specific homeobox 4 (Obox4) is critical for maintaining the intact nuclear membrane of the germinal vesicle (GV) in oocytes and for completing meiosis at the metaphase I–II (MI–MII) transition. This study further examines the molecular mechanisms of OBOX4 in regulating GV nuclear membrane breakdown. Maturation-promoting factor (MPF) and MAPK are normally inactive in GV stage oocytes but were activated prematurely in arrested GV stage oocytes by 3-isobutyl-1-metyl-xanthine (IBMX)in vitroafterObox4RNA interference (RNAi). Furthermore, signal transducer and activator of transcription 3 (STAT3) was significantly activated byObox4RNAi. We confirmed that thisObox4RNAi-induced premature STAT3 and MPF/MAPK activation at the GV stage provoked subsequent GV breakdown (GVBD) despite the opposing force of high cAMP in the IBMX-supplemented medium to maintain intact GV. When cumulus–oocyte complexes were exposed to interferon α (IFNA), a STAT3 activator, oocytes matured and cumulus cells expanded to resume nuclear maturation in IBMX-supplemented medium, suggesting that STAT3 activation is sufficient for stimulating the continuation of meiosis. Using Stattic, a specific STAT3 inhibitor, we confirmed that GVBD involves STAT3 activation inObox4-silenced oocytes. Based on these findings, we concluded that i)Obox4is an important upstream regulator of MPF/MAPK and STAT3 signaling, and ii)Obox4is a key regulator of the GV arrest mechanism in oocytes.

Sign in / Sign up

Export Citation Format

Share Document