Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes

2003 ◽  
Vol 179 (1) ◽  
pp. 25-34 ◽  
Author(s):  
M Shimada ◽  
J Ito ◽  
Y Yamashita ◽  
T Okazaki ◽  
N Isobe
Keyword(s):  
High Activity ◽  
Cell Function ◽  
Kinase Inhibitor ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Basic Medium ◽  
Phosphatidylinositol 3 Kinase ◽  
Germinal Vesicle Breakdown ◽  
Phosphatidylinositol 3

In this study, we investigated the mechanisms of protein kinase B (PKB) activation and its role in cumulus cells during in vitro meiotic resumption of porcine oocytes. PKB activity in cumulus cells was significantly decreased by 12 h cultivation of cumulus-oocyte complexes (COCs) in basic medium. However, the addition of phosphodiesterase inhibitors, hypoxanthine or 3-isobutyl-1-methylxanthine, maintained the level of PKB activity in cumulus cells at comparable with that in cumulus cells just after collection from their follicles. When COCs were cultured with phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, PKB activity was significantly decreased, and both caspase 3 activity and the proportion of apoptotic cells were significantly increased as compared with those in cumulus cells just after collection from their follicles. Moreover, the inhibitory effect of hypoxanthine on spontaneous meiotic resumption was overcome by addition of LY294002. On the other hand, markedly high activity of PKB and high intensity of the phosphorylated PKB band were observed in cumulus cells of COCs which were cultured with FSH. The addition of 20 microM LY294002 to FSH-containing medium induced an apoptosis of cumulus cells, whereas little apoptotic-positive signal was detected in COCs cultured with 5 microM LY294002 and FSH. However, the inhibitory effects of LY294002 on progesterone production by cumulus cells and germinal vesicle breakdown in oocytes reached a maximum at 5 microM. Thus, high activity of the PI 3-kinase-PKB pathway in cumulus cells plays an important role in FSH regulation of cell function. Judging from these results, it is estimated that PI 3-kinase in cumulus cells is required for both the suppression of spontaneous meiotic resumption and the induction of gonadotropin-stimulated meiotic resumption.

scholarly journals SIP/SHIP inhibits Xenopus oocyte maturation induced by insulin and phosphatidylinositol 3-kinase.

1997 ◽  
Vol 17 (5) ◽  
pp. 2559-2565 ◽  
Author(s):  
M Deuter-Reinhard ◽  
G Apell ◽  
D Pot ◽  
A Klippel ◽  
L T Williams ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Sh2 Domain ◽  
Mitogen Activated Protein Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Germinal Vesicle Breakdown ◽  
Inositol Phosphatase ◽  
Phosphatidylinositol 3

SIP (signaling inositol phosphatase) or SHIP (SH2-containing inositol phosphatase) is a recently identified SH2 domain-containing protein which has been implicated as an important signaling molecule. SIP/SHIP becomes tyrosine phosphorylated and binds the phosphotyrosine-binding domain of SHC in response to activation of hematopoietic cells. The signaling pathways and biological responses that may be regulated by SIP have not been demonstrated. SIP is a phosphatidylinositol- and inositol-polyphosphate 5-phosphatase with specificity in vitro for substrates phosphorylated at the 3' position. Phosphatidylinositol 3'-kinase (PI 3-kinase) is an enzyme which is involved in mitogenic signaling and whose phosphorylated lipid products are predicted to be substrates for SIP. We tested the hypothesis that SIP can modulate signaling by PI 3-kinase in vivo by injecting SIP cRNAs into Xenopus oocytes. SIP inhibited germinal vesicle breakdown (GVBD) induced by expression of a constitutively activated form of PI 3-kinase (p110*) and blocked GVBD induced by insulin. SIP had no effect on progesterone-induced GVBD. Catalytically inactive SIP had little effect on insulin- or PI 3-kinase-induced GVBD. Expression of SIP, but not catalytically inactive SIP, also blocked insulin-induced mitogen-activated protein kinase phosphorylation in oocytes. SIP specifically and markedly reduced the level of phosphatidylinositol (3,4,5) triphosphate [PtdIns(3,4,5)P3] generated in oocytes in response to insulin. These results demonstrate that a member of the phosphatidylinositol polyphosphate 5-phosphatase family can inhibit signaling in vivo. Further, our data suggest that the generation of PtdIns(3,4,5)P3 by PI 3-kinase is necessary for insulin-induced GVBD in Xenopus oocytes.

scholarly journals In Vitro Antimetastatic Effect of Phosphatidylinositol 3-Kinase Inhibitor ZSTK474 on Prostate Cancer PC3 Cells

2013 ◽  
Vol 14 (7) ◽  
pp. 13577-13591 ◽  
Author(s):  
Wennan Zhao ◽  
Wenzhi Guo ◽  
Qianxiang Zhou ◽  
Sheng-Nan Ma ◽  
Ran Wang ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Kinase Inhibitor ◽  
Phosphatidylinositol 3 Kinase ◽  
Antimetastatic Effect ◽  
Pc3 Cells ◽  
Phosphatidylinositol 3

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

IN VITRO METABOLISM OF THE PHOSPHATIDYLINOSITOL 3-KINASE INHIBITOR, WORTMANNIN, BY CARBONYL REDUCTASE

2004 ◽  
Vol 32 (5) ◽  
pp. 490-496 ◽  
Author(s):  
Julianne L. Holleran ◽  
Julien Fourcade ◽  
Merrill J. Egorin ◽  
Julie L. Eiseman ◽  
Robert A. Parise ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Carbonyl Reductase ◽  
In Vitro Metabolism ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

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).

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.

265 INHIBITION OF CLASS III PHOSPHATIDYLINOSITOL-3-KINASE, BY 3-METHYLADENINE, REVERSIBLY ARRESTS PORCINE OOCYTES AT GERMINAL VESICLE STAGE

2011 ◽  
Vol 23 (1) ◽  
pp. 230
Author(s):  
M. R. Park ◽  
M. K. Gupta ◽  
S. J. Uhm ◽  
S. T. Shin ◽  
Y. M. Han ◽  
...  
Keyword(s):  
Specific Inhibitor ◽  
Indirect Evidence ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Class Iii ◽  
Cleavage Rate ◽  
Chi Square ◽  
Meiotic Progression ◽  
Phosphatidylinositol 3

Phosphatidylinositol-3-kinases (PI3K) play pivotal roles in the meiotic progression of oocytes from metaphase I to metaphase II stage. This study evaluated the effect of 3-methyladenine (3MA), a specific inhibitor of Class III PI3K, on the meiotic progression of porcine oocytes. Immature porcine oocytes (n = 4744) retrieved from abattoir-derived oocytes were cultured in the absence or presence (10 mM) of 3MA for 22 h and evaluated for meiotic progression by florescent Hoechst 33342 staining. Data were analysed by chi-square test or ANOVA using SPSS software, and differences at P < 0.05 were considered significant. Results showed that 3MA treatment arrested the immature porcine oocytes at germinal vesicle (GV) stage in the presence (99.2 ± 0.8 v. 54.0 ± 10.1%) or absence (96.5 ± 1.8 v. 41.0 ± 17.6%) of cumulus cells. Furthermore, a significantly high proportion (60.9 ± 13.8%) of 3MA-treated oocytes acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (GV-II stage). When immature oocytes, arrested at GV stage for 22 h by 3MA, were further cultured for 22 h in the absence of 3MA, 96.1 ± 1.5% of oocytes reached metaphase II stage at 42 h of in vitro maturation and did not differ (P > 0.05) from nontreated control oocytes with respect to their ability to fertilize, cleave (74.1 ± 1.2 v. 72.7 ± 2.8%), and form blastocyst (15.4 ± 1.5 v. 12.7 ± 0.6%) upon IVF or parthenogenetic activation (cleavage rate: 89.8 ± 1.7 v. 84.6 ± 5.1%; blastocyst rate: 44.3 ± 12.4 v. 45.1 ± 7.6%). These data suggest that 3MA reversibly blocks and synchronizes the meiotic progression of porcine oocytes at GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocytes beyond GV stage. This work was supported by grants (Code #200901FHT010305191 and #20070401034017) from BioGreen 21 program of RDA, Republic of Korea.

scholarly journals Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

1996 ◽  
Vol 7 (3) ◽  
pp. 355-367 ◽  
Author(s):  
D J Spiro ◽  
W Boll ◽  
T Kirchhausen ◽  
M Wessling-Resnick
Keyword(s):  
Transferrin Receptor ◽  
Kinase Inhibitor ◽  
Morphological Changes ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Phosphatidylinositol 3 Kinase ◽  
Vitro Assay ◽  
Phosphatidylinositol 3

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

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