MAPK/ERK kinase (MEK) signalling is required for resumption of meiosis in cultured cumulus-enclosed pig oocytes

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 7-16 ◽  
Author(s):  
B. Meinecke ◽  
C. Krischek
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Mek Inhibitor ◽  
Free Medium ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Mitogen Activated Protein ◽  
Drug Free ◽  
Medium Culture

Resumption of meiosis of mammalian oocytes is facilitated by the maturation promoting factor (MPF) and accompanied by activation of mitogen activated protein kinases (MAPK) which are phosphorylated by the MAPK kinase (MEK). In this study we examined the effects of PD 98059, which inhibits the activity of MEK, on in vitro maturation of pig oocytes. Cumulus-oocyte complexes (COCs) were cultured in the presence or absence of the drug (50 μM) for various time periods. To elucidate the influence of cumulus cells, COCs were first cultured in inhibitor-free medium, subsequently denuded, and incubated further in PD 98059 supplemented medium. Reversibility of drug action as tested following PD 98059 treatment of COCs by transferring them to drug-free medium. Culture of COCs in medium supplemented with PD 98059 prevents resumption of nuclear maturation in the majority of COCs. This inhibition was reversible and accompanied by a non-activation of both MAP and MPF. Addition of the MEK inhibitor to extracts of in vitro matured oocytes revealed that the kinase activities were not directly influenced by the inhibitor, suggesting a link between MAP and MPF kinases. Preincubation of COCs in inhibitor-free medium for 6 h followed by further culture of COCs or denuded oocytes in the presence of PD 98059 for various periods resulted in elevated MAP and MPF kinase activities, indicating an early and transient MEK signalling in the oocyte itself. These results support the idea that MAP and MPF are involved in the induction of germinal vesicle breakdown in porcine oocytes.

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.

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

83 VITRIFICATION OF IMMATURE AND IN VITRO-MATURED HORSE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 149 ◽  
Author(s):  
L. Bogliolo ◽  
F. Ariu ◽  
I. Rosati ◽  
M. T. Zedda ◽  
S. Pau ◽  
...  
Keyword(s):  
Survival Rate ◽  
Ethylene Glycol ◽  
Survival Rates ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hoechst 33342 ◽  
Nuclear Maturation ◽  
And Control ◽  
Cryopreservation Protocol

Few attempts have been carried out to cryopreserve equine oocytes, and an effective cryopreservation protocol is not defined yet. Studies were conducted to compare the viability of immature and in vitro-matured horse oocytes vitrified by the minimal volume cooling (MVC) cryotop vitrification method (Kuwayama et al. 2005 Reprod. BioMed. Online 11, 300–308). Oocytes were recovered from slaughterhouse ovaries and divided, on the basis of the morphology of cumulus cells, into cumulus-expanded (CE) and cumulus-compacted (CC) oocytes. Groups of CC and CE oocytes were vitrified immediately after recovery [germinal vesicle (GV) stage] or matured in vitro (IVM) and cryopreserved at the MII stage as follows: oocytes were incubated 30 min in TCM-199 + 20% FCS + 10% ethylene glycol (EG) + 10% DMSO, followed by 20 min in TCM-199 + 20% FCS + 20% EG + 20% DMSO + 0.25 M sucrose, loaded in cryotops (2 µL), and plunged into liquid nitrogen. Warming was performed at 38.5°C by washing the oocytes in TCM-199 + 20% FCS with decreasing sucrose concentrations (1.25 M, 0.62 M, 0.31 M). After warming oocytes cryopreserved at the GV stage were matured in vitro for 24 h (CE) or 36 h (CC) in TCM-199 + 10% FCS + FSH, LH each at (0.1 UI/mL) + cysteamine, fixed, and stained with glycerol-Hoechst 33342 to assess nuclear maturation. Oocytes vitrified at the MII stage were in vitro cultured for 2 h to evaluate their morphological survival on the basis of the presence of an intact zona pellucida and membrane. Nonvitrified oocytes undergoing the same maturation protocol were used as controls. Results (Table 1) indicated that the survival rate of oocytes vitrified at the GV stage, after IVM, was similar between CE and CC oocytes (43.6% vs 42.6%). Significantly (P < 0.01) higher numbers of vitrified CE MII oocytes (52.9%) survived, compared to CC (34.8%), after 2-h culture. The percentages of viable MII oocytes from CE and CC GV vitrified oocytes were 43.6% and 40.9% respectively and were comparable to those from vitrified MII oocytes (CE, 52.9%; CC, 34.8%) and control oocytes (CE, 56.4%; CC, 53.3%). In conclusion, the results of this study showed that vitrification by the MCV Cryotop method of horse oocytes at either the GV or the MII stage allows a similar number of viable mature oocytes to be recovered. Table 1. Maturation and survival rates of immature and mature equine oocytes vitrified by the MCV Cryotop method

336 MEIOTIC DEVELOPMENT AND CORTICAL GRANULES DISTRIBUTION IN CANINE OOCYTES DURING IN VITRO MATURATION

2010 ◽  
Vol 22 (1) ◽  
pp. 324 ◽  
Author(s):  
M. De los Reyes ◽  
D. Luna ◽  
J. Palomino
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Fluorescein Isothiocyanate ◽  
Cumulus Cells ◽  
Homogeneous Distribution ◽  
Cortical Granules ◽  
Dapi Staining ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Low development of IVM canine oocytes could be in part attributed to an impaired cytoplasmic maturation. In mammalian oocytes, migration and the redistribution of cortical granules (CGs) around the periphery of the oocyte contribute to the inhibition of polyspermy and it is an important criterion to evaluate cytoplasmic maturation. The state of nuclear maturation and the distribution of CGs were evaluated in canine oocytes cultured for different periods in order to compare the synchrony of nuclear and cytoplasmic maturation during in vitro maturation. Bitch ovaries at different stages of the estrous cycle were obtained following ovariectomy. COCs with compact cumulus cells showing a homogeneous cytoplasm were selected for experiments. Thirty-six COCs were processed at immature stage, placed in PBS medium until evaluation. A total of 275 COCs were matured in vitro for 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM Hepes, 10% FCS, 0.25 mM pyruvate, 10 IU mL-1 of hCG, 300 IU mL-1 penicillin, and 20 mg mL-1 streptomycin, at 38.5°C and 5% CO2. At each culture period, the oocytes were stained with Lens culinaris agglutinin (LCA), labeled with fluorescein isothiocyanate, and the CGs distributions were examined under a fluorescent microscope. The nuclear status of the denuded oocytes was determined by DAPI staining under a fluorescence microscope. For each treatment, at least four replicates were performed and the data was analyzed by ANOVA using Tukey’s test to determine the differences P < 0.05. Three types of CGs distribution were distinguished during canine oocyte maturation: (1) homogeneous distribution throughout the cytoplasm including the cortex; (2) heterogeneous (clusters) within the cytoplasm and (3) densely distributed beneath the oolemma. Nuclear stages were classified as immature or germinal vesicle (GV) stage; resumption of meiosis or germinal vesicle break down (GVBD); metaphase I to telophase I (MI toTel I); and mature or second metaphase (MII). The distribution patterns of GCs were different (P < 0.05) among oocytes cultured for different periods and the nuclear maturation status also differed between oocytes cultured for different intervals (P < 0.05). Most (>84%) of the immature oocytes at GV showed a uniform distribution of CGs throughout the cytoplasm. At 48 h of culture, CGs distribution was mainly Type 2 (25%) and 3 (61%) and the oocytes were at GVBD (33%) and MI-Tel I (33%) stages. Most nuclei of the type 3 oocytes were in the MI (40%) and MII (11%) stages, corresponding to those oocytes matured for 72 (88%) or 96 h (71%). These results indicate that canine oocytes migrate to the cortex during IVM and this process is not finished before 72 h of culture. In addition, although the re-distribution of the CGs occurred in parallel with nuclear maturation, the oocytes cannot always proceed to the MII stage; however, in such oocytes the CGs are distributed beneath the oolemma. Supported by Grant FONDECYT 1080618.

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.

Time course of the meiotic arrest in sheep cumulus–oocyte complexes treated with roscovitine

Zygote ◽  
2015 ◽  
Vol 24 (2) ◽  
pp. 310-318 ◽  
Author(s):  
Letícia Ferrari Crocomo ◽  
Wolff Camargo Marques Filho ◽  
Camila Louise Ackermann ◽  
Daniela Martins Paschoal ◽  
Midyan Daroz Guastali ◽  
...  
Keyword(s):  
Exposure Time ◽  
Cell Expansion ◽  
Time Course ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Nuclear Maturation ◽  
Maturation Medium

SummaryTemporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus–oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

scholarly journals Cyclic Adenosine 3′,5′-Monophosphate-Dependent Activation of Mitogen-Activated Protein Kinase in Cumulus Cells Is Essential for Germinal Vesicle Breakdown of Porcine Cumulus-Enclosed Oocytes

Endocrinology ◽  
2005 ◽  
Vol 146 (10) ◽  
pp. 4437-4444 ◽  
Author(s):  
Cheng-Guang Liang ◽  
Li-Jun Huo ◽  
Zhi-Sheng Zhong ◽  
Da-Yuan Chen ◽  
Heide Schatten ◽  
...  
Keyword(s):  
Mapk Pathway ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Mitogen Activated Protein Kinase ◽  
Pde4 Inhibitor ◽  
Germinal Vesicle Breakdown ◽  
Germinal Vesicles ◽  
Mitogen Activated Protein ◽  
Culture Models ◽  
Cell Type Specific

MAPK plays an important role during meiotic maturation in mammalian oocytes, whereas the necessity of MAPK during meiotic resumption in porcine oocytes is still controversial. Here, by applying the method of ultracentrifugation to move the opaque lipid droplets to the edge of the oocyte, therefore allowing clear visualization of porcine germinal vesicles, oocytes just before germinal vesicle breakdown (GVBD) and those that had just undergone GVBD were selected for the assay of MAPK activation. Our results showed that phosphorylation of MAPK in oocytes occurred after GVBD in all three different culture models: spontaneous maturation model, inhibition-induction maturation model, and normal maturation model. Moreover, we found that activation of MAPK in cumulus cells but not in oocytes was essential for GVBD in cumulus-enclosed oocytes. Then the cross-talk between cAMP and MAPK in cumulus cells was investigated by using cell-type-specific phosphodiesterase (PDE) isoenzyme inhibitors. Our results showed that PDE3 subtype existed in oocytes, whereas PDE4 subtype existed in cumulus cells. PDE3 inhibitor prevented meiotic resumption of oocytes, whereas PDE4 inhibitor enhanced the ability of FSH or forskolin to activate MAPK in cumulus cells. We propose that increased cAMP resulting from inhibition of PDE3 in oocytes blocks GVBD, whereas increased cAMP resulting from inhibition of PDE4 activates MAPK pathway in cumulus cells, which is essential for GVBD induction.

scholarly journals 78 SURVIVAL OF PORCINE OOCYTES AT GERMINAL VESICLE STAGE AFTER VITRIFICATION WITH OPEN PULLED STRAW METHOD

2005 ◽  
Vol 17 (2) ◽  
pp. 189 ◽  
Author(s):  
A. Bali Papp ◽  
T. Somfai ◽  
E. Varga ◽  
M. Marosán
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Penicillin G ◽  
National Committee ◽  
Ministry Of Education ◽  
Germinal Vesicle Breakdown ◽  
Maturation Medium ◽  
Maturation Rate ◽  
Nuclear Stage

The present study was performed to assess the survival of immature denuded or cumulus-covered porcine oocytes (COCs). Immature porcine oocytes were collected from 2–6 mm follicles of slaughterhouse ovaries and subjected to open pulled straw (OPS) vitrification, according to the method of Vajta et al. (1998 Mol Reprod. Dev. 51, 53–58). After vitrification, oocytes were matured in vitro for 48 h at 39°C, 5% CO2 in air. The maturation medium was TCM199 supplemented with 10% pig follicular fluid, 1.25 mM L-glutamine, 0.9 mM Na pyruvate, 150 μM cysteamine, 0.1 mg/mL streptomycin sulfate, 100 IU/mL PG penicillin g potassium, 10 IU/mL PMSG, and 25 IU/mL hCG. After IVM, to assess nuclear stage, all oocytes were fixed with acetic acid–alcohol (1:3) for at least three days and then stained with 0.1% orcein and examined under a phase-contrast microscope at 100× magnification. All data were analyzed by χ2 test (P < 0.05). Immediately after collection, all oocytes were at the germinal vesicle (GV) stage with an intact GV membrane. After vitrification, significantly fewer oocytes had normal morphology (intact plasma membrane) in the denuded and COC groups (4.7% and 8.5%, respectively) than did the denuded and COC control groups (95% and 92%, respectively). By the end of IVM, significantly fewer oocytes were surrounded by expanded cumulus after vitrification of COCs than were the COC controls (28.1% and 63.5%, respectively). After IVM, more of the COC control oocytes underwent germinal vesicle breakdown than did the denuded controls (95% and 78.2%, respectively); the rate of MII oocytes was higher for the COC controls than for the denuded controls (80% and 54.5%, respectively). After vitrification, the number of oocytes that underwent GVBD was significantly less for both the denuded and the COC groups (2.0% and 7.0%, respectively); the percentage of oocytes that reached MII was also lower (0.64% and 2.78%, respectively). Most of the vitrified oocytes had a damaged GV with disrupted membrane and cluster-like or scattered chromatin in both the denuded and the COC groups (96.4% and 90.7%, respectively). These data suggest that vitrification of cumulus-enclosed immature porcine oocytes is preferable compared to vitrification of denuded ones. Loss of cumulus cells compromises competence of oocytes to resume meiosis, which might result in a lower maturation rate after IVM. This research was supported by the grants of the Hungarian Scientific Research Fund (T 031758), the Hungarian National Committee of the Technical Development at the Ministry of Education (00796/2003), and the Ministry of Education (OM-KMUFA; BIO-00086/2002).

344 PENTOSE PHOSPHATE PATHWAY ACTIVITY CONTROLS NUCLEAR MATURATION OF PORCINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 279 ◽  
Author(s):  
L. Tubman ◽  
A. Peter ◽  
R. Krisher
Keyword(s):  
Pentose Phosphate Pathway ◽  
Control Level ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Germinal Vesicle ◽  
Pentose Phosphate ◽  
Meiotic Stage ◽  
Germinal Vesicle Breakdown ◽  
Pathway Activity ◽  
Nuclear Maturation

Diphenyleneiodonium (DPI), an inhibitor of the pentose phosphate pathway (PPP), arrests nuclear maturation of porcine oocytes. This inhibition is reversed using products or cofactors of PPP such as nicotinamide adenine dinucleotide phosphate (NADP), phosphoribose diphosphate (PRPP), and ribose-5-phosphate (R5P). The objective of this study was to determine the relationship between DPI-mediated meiotic inhibition, reversal of this inhibition, and metabolism of in vitro-matured (IVM) porcine oocytes. Oocytes were aspirated, searched, and selected in the presence of DPI, with the exception of control oocytes. Oocytes were then matured in one of five treatments for 40 h in 7% CO2 in air at 39°C in defined Purdue Porcine Medium for maturation (PPMmat). Treatments included control, 50 nM DPI (DPI), DPI + 5 mM NADP (NADP), DPI + 12.5 mM PRPP (PRPP), and DPI + 10 mM R5P (R5P). Following IVM, oocytes were denuded by vortexing. Glycolysis and PPP activities were measured in 4 μL hanging drops containing labeled glucose (0.0125 mM 5-3H glucose and 0.482 mM 1-14C glucose, respectively) for 3 h in 6% CO2. Oocytes were then individually fixed in a 3:2:1 solution of ethanol:acetic acid:chloroform and stained with aceto-orcein for determination of meiotic stage (germinal vesicle = 1 through metaphase II = 7). Data were analyzed using one-way ANOVA. The use of DPI inhibited PPP and nuclear maturation; additionally glycolysis was decreased by DPI compared to control. Addition of NADP and PRPP increased both metabolic pathways and nuclear maturation compared to DPI. R5P restored glycolysis and nuclear maturation to control levels, and PPP to above the control level. There were no significant differences among meiotic stages relative to glycolytic activity. PPP activity was significantly different (values with different superscripts; P < 0.05) among oocytes of different meiotic stages (germinal vesicle = 0.24 ± 0.03ad, germinal vesicle breakdown = 0.40 ± 0.05bcde, condensed chromatin = 0.44 ± 0.05bcd, metaphase I = 0.45 ± 0.12abcd, anaphase = 0.76 ± 0.50abcde, telophase = 0.92 ± 0.17be, metaphase II = 0.74 ± 0.08be). Percentages of oocytes reaching MII were 43.48 (control), 2.08 (DPI), 28.30 (NADP), 18.18 (PRPP), and 46.94 (R5P). These results demonstrate that the PPP is a critical control mechanism for nuclear maturation of porcine oocytes, as inhibition of this metabolic pathway resulted in arrest of nuclear maturation. Addition of PPP cofactors or end products to the arresting medium led to reversal of inhibition as demonstrated by restoration of PPP activity resulting in nuclear maturation. Table 1. Meiotic stage, glycolysis, and pentose phosphate pathway activity after in vitro maturation of porcine oocytes

scholarly journals 281 MITOGEN-ACTIVATED PROTEIN KINASE IN PORCINE CUMULUS CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 291
Author(s):  
S. Ebeling ◽  
C. Boesebeck ◽  
B. Meinecke
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Active Form ◽  
Cumulus Cells ◽  
Mitogen Activated Protein Kinase ◽  
Santa Cruz ◽  
High Concentration ◽  
Germinal Vesicle Breakdown ◽  
Mitogen Activated Protein

Mitogen-activated protein kinase (MAPK) is involved in many signal processes within eukaryotic organisms. Its active form is phosphorylated. For meiotic resumption in oocytes the MAPK cascade plays a central role, because it participates in the transfer of the extracellular gonadotropin signal into the nucleus. In pigs it could be shown that for a gonadotropin-induced germinal vesicle breakdown (GVBD), an activation of MAPK in oocytes is not essential, but in the surrounding cumulus cells the MAPK has to be phosphorylated (Ohashi et al. 2003 Biol. Reprod. 68, 604–609). Because cumulus cells are very important for signal transfer, the present investigation dealt with the relevance of porcine cumulus cells and the phosphorylation of MAPK for the resumption of meiosis. Oocytes of slaughtered pigs were collected and cultured (medium: TCM 199, insulin, l-glutamine, gentamycin, 20% (v/v) FCS, and with or without 2.5 μg/mL FSH and 5.0 μg/mL LH). The proteins of isolated cumulus cells and oocytes were separated by gel electrophoresis (cumulus cells of 10 cumulus-oocyte complexes and 40 oocytes per lane, respectively) followed by an immunoblot with antibodies against MAPK and p90rsk (ERK 1 (sc-94) and Rsk-1 (sc-231), respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Alternatively the nuclear maturation was determined by orceine staining. The following results were achieved: The phosphorylation of MAPK in cumulus cells began very early during the in vitro maturation period. This was demonstrated already after 0.5 h unlike in oocytes where phosphorylation of MAPK does not occur until 18 h. The phosphorylation in cumulus cells occurs both in the presence and in the absence of FSH/LH, but without FSH/LH almost no GVBD occurs (after 26 h IVM: 86.9% GV oocytes, n = 59). The phosphorylation in the absence of gonadotropins could be caused by components of FCS, but with an exchange against polyvinylpyrrolidone (0.3%), the phosphorylation without FSH/LH still existed. The specificity was examined with the MAPK kinase inhibitor U0126. A concentration of 10 μM U0126 prevented GVBD and phosphorylation of MAPK in oocytes. However, in cumulus cells the phosphorylation of MAPK was reduced only minimally. In the presence of 50 μM U0126, a distinct decrease was observed during the first hours of maturation. But after 26 h phosphorylated MAPK appeared in cumulus cells despite the high concentration of U0126. The p90rsk is an important substrate of MAPK, which is phosphorylated by activated MAPK in oocytes. In our investigations we could detect only unphosphorylated forms of p90rsk in the cumulus cells. It seems that there are different ways for phosphorylation of MAPK to occur in cumulus cells, but they do not have the same consequences. The phosphorylation of MAPK in cumulus cells is necessary for a gonadotropin induced meiotic resumption, but phosphorylation does not always lead to GVBD. Furthermore, the p90rsk appears not to have the same importance as a substrate of MAPK in cumulus cells as in oocytes.

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