The kinase inhibitor indirubin-3'-oxime prevents germinal vesicle breakdown and reduces parthenogenetic development of pig oocytes

Oocytes undergo spontaneous germinal vesicle breakdown (GVBD) after being released from the follicular environment; this potentially prevents manipulation of the oocyte at the germinal vesicle (GV) stage. The objectives of this study were to investigate the effects of indirubin, a potent cdc2 kinase inhibitor, on GVBD and microtubular structure of porcine oocytes. Cumulus-oocyte-complexes (COCs) were collected from abattoir-derived ovaries and were randomly allocated to different concentrations of indirubin treatments (0, 10, 25, 50, and 100 microM in Experiment 1 and 0, 50, 75, and 100 microM in Experiment 2) during 44 h of IVM. The influences on the GVBD, microtubules, and maturation rates were evaluated using epifluorescence microscopy. The percentages of oocytes remaining at the GV stage were 0, 16, 26, 69, and 85% for oocytes treated with 0, 10, 25, 50, and 100 microM of indirubin, respectively, which differed among treatment groups (P<0.05). However, there were no significant differences between the oocytes treated with 75 and 100 microM (79 and 81%). The cytoplasmic microtubules were fragmented in oocytes maintained at the GV stage and the chromatin became condensed or aggregated. When COCs were incubated with indirubin (50-75 microM) for 22 h and then transferred to maturation medium for 44 h (Experiments 3-5), the percentages of oocytes reaching the metaphase II stage were generally higher than when the COCs were cultured in the presence of the drug for 44 h (62-65% versus 44-46%). However, the parthenogenetic development of the oocytes in Experiment 6 was reduced significantly in drug-treated oocytes. In summary, treatment with 50-75 microM of indirubin effectively prevented GVBD in porcine oocytes, but the developmental competence of the oocytes was compromised.     

Interactive effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes

The objective of this article was to study the effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes. Goat oocytes were cultured at different temperatures in medium containing different concentrations of ROS, and at the end of culture, oocytes were either matured or processed for light/confocal microscopy. The matured oocytes were activated chemically or fertilized in vitro for embryo development. Meiotic arrest was successfully maintained for 24 hr with 0, 50, and 200 microM ROS at 5, 20, and 38.5 degrees C, respectively. Following chemical activation, morulae/blastocysts (M/B) rates similar to untreated oocytes were obtained in oocytes that had been inhibited for 24 hr at 5 degrees C without ROS (Protocol 5C) or at 20 degrees C with 50 microM ROS (Protocol 20C) or for 8 hr at 38.5 degrees C with 200 microM ROS (Protocol 8 hr), but no blastulation was observed after oocytes were inhibited at 38.5 degrees C with 200 microM ROS for 24 hr. Following fertilization, however, while M/B rates similar to controls were achieved in oocytes treated with protocols 5C and 20C, few oocytes inhibited with Protocol 8 hr developed into morulae, due to a high incidence of polyspermy. Changes in GV chromatin configuration were not observed after inhibition with Protocol 5C, but were apparent after inhibition with protocols 20C and 8 hr, leading to a precocious germinal vesicle breakdown (GVBD) during subsequent maturation. Cortical granule (CG) migration and the formation of microtubule organizing centers occurred during inhibition and were more obvious in the absence of ROS. Significantly more oocytes inhibited by protocols 5C and 20C than by Protocol 8 hr completed CG migration after maturation. In conclusion, goat oocytes were tolerant to chilling and culture at lower temperatures with less ROS was better than culture at higher temperatures with more ROS for oocyte GVBD inhibition.     

Effect of Cleome arabica leaf extract, rutin and quercetin on soybean lipoxygenase activity and on generation of inflammatory eicosanoids by human neutrophils

The effects of Cleome arabica leaf extract, rutin and quercetin on soybean lipoxygenase (Lox) activity and on calcium ionophore (A23187)-stimulated generation of the leukotriene B4 and prostaglandin E2 by human neutrophils were examined. The extract (25 microg/ml), rutin (25 microM) and quercetin (25 microM) inhibited LTB4 synthesis at all concentrations of A23187 used. The extract at 1-100 microg/ml and rutin at 1-100 microM inhibited LTB4 generation by neutrophils stimulated with 1 microM A23187 by about 50%. PGE2 production in response to different concentrations of A23187 was affected in a biphasic manner by the extract and rutin. Quercetin at 1-100 microM caused concentration-dependent inhibition of LTB4 and PGE2 production. The extract, rutin and quercetin caused concentration-dependent inhibition of soybean Lox activity. These results indicate that rutin, quercetin and an extract of C. arabica containing these compounds inhibit Lox activity, consequently decreasing LTB4 production. Thus, these compounds or extracts containing them may be beneficial for the treatment of inflammatory conditions, particularly those characterised by excessive leukotriene generation.     

Cytoplasmic reorganization during the resumption of meiosis in cultured preovulatory rat oocytes

Changes in organelle topography and microtubule configuration have been studied during the resumption and progression of meiosis in cultured preovulatory rat oocytes. Germinal vesicle breakdown (GVBD) was reversibly inhibited by dibutyryl cAMP (DcAMP) or nocodazole, a microtubule-disrupting agent. The microtubule stabilizing agent taxol did not inhibit GVBD, but did impair further maturation. The migration of acidic organelles and chromatin in living oocytes was analyzed using the vital stains acridine orange and Hoechst 33258, respectively. Germinal vesicle stage oocytes undergo perinuclear aggregation of acidic organelles during GVBD and these organelles subsequently disperse into the cell cortex as the first meiotic spindle migrates to the oocyte periphery. DcAMP and nocodazole block the perinuclear aggregation of acidic organelles, whereas, in taxol-treated oocytes, organelle aggregation and GVBD occur but the dispersion of acidic organelles was arrested. Dose-response studies on the effects of nocodazole showed that GVBD was generally retarded and that a 50% inhibition of GVBD was achieved at concentrations in excess of 1.0 microM. Concentrations of taxol at 10 microM or above effectively inhibited both chromatin condensation and meiotic spindle formation. Indirect immunofluorescence microscopy with anti-tubulin antibodies revealed dissolution of microtubules with 1.0 microM nocodazole. Taxol had little effect on microtubule organization in germinal vesicle or chromatin condensation stage oocytes; however, when oocytes that had formed first meiotic spindles were treated with taxol, numerous microtubule asters appeared which were preferentially associated with the oocyte cortex. The changes in organelle topography, microtubule configuration, and drug sensitivity are discussed with respect to the regulation of cytoplasmic reorganization during the meiotic maturation of rat preovulatory oocytes.     

Effect of 6-dimethylaminopurine on germinal vesicle breakdown of bovine oocytes

The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%, 120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6-DMAP were used (100-500 microM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis-metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximide-supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.     

Two phases of calcium requirement during starfish meiotic maturation

During meiosis in oocytes of the starfish, Asterina pectinifera, a Ca(2+) transient has been observed. To clarify the role of Ca(2+) during oocyte maturation in starfish, an intracellular Ca(2+) blocker, TMB-8, was applied. The oocyte maturation induced by 1-methyladenine (1-MA) was blocked by 100 microM TMB-8. Reinitiation of meiosis with germinal vesicle breakdown (GVBD) and the following chromosome condensation did not take place. Maturation-promoting factor (MPF) activity did not increase and GVBD and chromosome condensation did not occur. Ca(2+) transient observed immediately after 1-MA application in control oocytes was also blocked by TMB-8. When calyculin A, which activate the MPF directly, was applied to the oocytes instead of 1-MA in seawater containing 100 microM TMB-8, GVBD and chromosome condensation were blocked. Cytoplasmic transplantation studies confirmed that MPF was activated, although TMB-8 blocked GVBD. These results show that TMB-8 blocked the increase of MPF activity induced by 1-MA and the process of active MPF inducing GVBD and subsequent chromosome condensation. Together with the above phenomena, it is conceivable that there are two phases of Ca(2+) requirement during starfish oocyte maturation. These are the activation of MPF, moreover, GVBD, and the subsequent chromosome condensation.     

Modulation of oocyte maturation by cyclic adenosine 3', 5'-pyrophosphate

The germinal vesicle (GV) of follicle-enclosed oocytes in mammals remains arrested at the dictyate state of meiosis. Upon releasing the oocytes from the follicles, the meiotic process resumes, leading to dissolution of the GV (GVBD), suggesting that factors in the follicular fluid sustain the meiotic arrest of oocytes. In the present study the spontaneous resumption of meiosis was blocked by the addition of cyclic adenosine 3', 5'-pyrophosphate (cAPP) plus dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP), at final concentrations of 25 and 50 microM, respectively. These compounds were ineffective when added separately at these concentrations. None of the other related compounds tested with dbcAMP blocked GVBD. Bovine follicular fluid (BFF) was analyzed for inhibitors of GVBD. BFF was extracted with 70% ethanol and the ethanolic extract chromatographed on Dowex 1-X8 column. The fraction eluted with 0.1 N HCl markedly inhibited GVBD of isolated mouse oocytes in combination with dbcAMP. The active BFF substance and cAPP block spontaneous GVBD of mouse oocytes and may be related substances. The present study supports the thesis that meiotic arrest at the dictyate stage in oocytes is sustained by factors present in follicular fluid and may act in association with cAMP.     

Involvement of protein kinase C in the regulation of oocyte maturation in amphibians (Rana dybowskii)

Ovarian oocytes of Rana dybowskii, isolated early in the hibernation period (late autumn), failed to mature, i.e., germinal vesicle breakdown (GVBD), in response to progesterone during in vitro follicle culture. Oocytes collected during the middle hibernation period matured in response to progesterone, whereas those collected late during the hibernation period (close to the breeding season) underwent spontaneous maturation without added hormone (Kwon et al., '89). The maturational response (GVBD) of oocytes, collected at the three stages of hibernation, to protein kinase C (PKC) activation was investigated and compared to that of progesterone stimulation. A phorbol ester, phorbol 12-myristate 13-acetate (TPA) was used for PKC activation. TPA addition to cultured follicles collected during the early or middle period of hibernation induced oocyte GVBD. The incidence of maturation (% GVBD) induced by TPA varied markedly between animals. TPA (10 microM) induced oocyte maturation in the presence or absence of follicle cells. The time course of the TPA-induced maturation was similar to that of progesterone-stimulated maturation (ED50, 7-9 h). TPA also accelerated the onset of maturation of the follicular oocytes exhibiting spontaneous in vitro maturation. Both TPA- and progesterone-stimulated maturation was blocked by treatment with cycloheximide (1 microgram/2 ml), forskolin (9 microM) (an adenylate cyclase stimulator), and verapamil (0.27 mM) (a calcium transport blocker). Treatment of oocytes with a calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (100 microM) or a PKC inactivator 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) (50 microM) likewise suppressed TPA- or progesterone-induced maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C activity, protein phosphorylation and germinal vesicle breakdown in Spisula oocytes

To test the possible role of protein kinase C (C-kinase) in regulating germinal vesicle breakdown (GVBD) in Spisula oocytes, we studied the effects of phorbol esters and antagonists of C-kinase on GVBD and protein phosphorylation. Responses to these agents were compared to those elicited by fertilization or increased extracellular K+. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent agonist of C-kinase, elicited GVBD with half-maximal stimulation at 20 nM. By contrast, 4 alpha-phorbol-12,13-didecanoate, a phorbol ester which does not stimulate C-kinase, did not trigger GVBD. TPA accelerated GVBD when induced by excess K+, but it did not affect the time course of the process when initiated by fertilization. Three structurally different antagonists of C-kinase (W-7, H-7, and retinol) all blocked GVBD when induced by fertilization or TPA. When oocytes were preincubated with [32P]orthophosphate and then stimulated to undergo GVBD by fertilization, TPA, or 45 mM K+, protein phosphorylation was greatly increased, especially for a polypeptide(s) of about 45 kDa. Phosphorylation increased prior to GVBD. Retinol inhibited phosphorylation in activated eggs. C-kinase activity was demonstrated in oocyte extracts. These results strongly suggest that protein phosphorylation by C-kinase is involved in the pathway that regulates GVBD in Spisula oocytes.     

Possible involvement of calmodulin in maturation and activation of Chaetopterus eggs

We report the isolation of calmodulin from oocytes of Chaetopterus pergamentaceus. The identification of this protein is based on (1) activation of beef heart cAMP phosphodiesterase, (2) heat stability, (3) sensitivity to chlorpromazine, and (4) electrophoretic mobility identical to that of porcine brain calmodulin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of either Ca2+ or EGTA. We treated oocytes with chlorpromazine and W-7 to investigate the involvement of calmodulin in meiosis initiation and egg activation. Very low concentrations of chlorpromazine inhibited germinal vesicle breakdown (GVBD). This effect was shown to be dependent upon bright indirect light, since the drug was much less effective at GVBD inhibition under conditions of very low illumination. Higher concentrations of chlorpromazine and W-7 (100 microM) inhibited GVBD and activated eggs with intact germinal vesicles as determined by fertilization envelope formation and the onset of ameboid activity. Neither egg activation nor inhibition of calmodulin stimulation of phosphodiesterase activity in vitro was affected by light. These results are consistent with a role for calmodulin in egg activation and GVBD, but suggest that chlorpromazine in bright light may prevent GVBD by some mechanism other than calmodulin inhibition.     

Effect of cycloheximide upon maturation of bovine oocytes

Germinal vesicle breakdown (GVBD) of bovine oocytes was completely blocked by cycloheximide added to culture medium at concentrations of 1-20 micrograms/ml. Nevertheless, under such conditions a certain degree of chromatin condensation inside the germinal vesicle was observed. The inhibitory effect was not influenced by the presence or absence of cumulus cells and was fully reversible; but the process of GVBD was then significantly accelerated. The critical period in which the proteins necessary for GVBD are synthesized lasts approximately the first 5 h of culture. When germinal vesicle-arrested oocytes are fused to maturing bovine oocytes containing condensed chromosomes, GVBD of immature oocytes occurs within 3 h, even in the presence of cycloheximide. In the mouse, GVBD cannot be inhibited by protein synthesis inhibitors. When immature mouse oocytes are fused with immature bovine oocytes and the giant cells are then cultured in cycloheximide-supplemented medium, both GVs are observed, or only mouse GVBD occurs in common cytoplasm after 8 h of culture. We conclude that protein synthesis is necessary for GVBD of bovine oocytes. Our results also suggest that maturation-promoting factor (MPF) is not autocatalytically amplified in mammalian oocytes.     

The effect of quercetin on cell cycle progression and growth of human gastric cancer cells

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. We examined the effects on cell growth of human malignant cells derived from the gastrointestinal tract and on cell cycle progression. Quercetin markedly inhibited the growth of human gastric cancer cells and the IC50 value was 32-55 microM. DNA synthesis was suppressed to 14% of the control level by the treatment with 70 microM quercetin for 2 days. Furthermore, quercetin blocked cell progression from the G1 to the S phase.     

Effect of cycloheximide on nuclear maturation of pig and mouse oocytes

Culture of mouse oocytes in medium with 1 or 100 micrograms cycloheximide/ml did not prevent germinal vesicle breakdown (GVBD). In contrast, GVBD in pig oocytes was absolutely blocked at concentrations of 1, 5, 10, 50 and 100 micrograms cycloheximide/ml, respectively. The inhibition of GVBD was not influenced by the presence or absence of cumulus cells and it was fully reversible. When cycloheximide treatment (5 micrograms/ml) was given after preincubation for 6, 12 and 16 h, GVBD occurred in 15, 46 and 75% of oocytes, respectively. It is concluded that proteins important for GVBD of pig oocytes were present in sufficient amounts at about 12 h of culture. The fusion of pig oocytes in metaphase I to oocytes with an intact germinal vesicle revealed that cycloheximide did not inhibit GVBD induced by maturing ooplasm. Therefore, induction of prematurely condensed chromosomes by the maturing ooplasm did not require protein synthesis. However, continuous protein synthesis was necessary to maintain metaphase I and prematurely condensed chromosomes in a typical configuration.     

Quercetin selectively inhibits insulin receptor function in vitro and the bioresponses of insulin and insulinomimetic agents in rat adipocytes.

We report here that quercetin, a naturally occurring bioflavonoid, is an effective blocker of insulin receptor tyrosine kinase-catalyzed phosphorylation of exogenous substrate. The ID50 was estimated to be 2 +/- 0.2 microM in cell-free experiments, using a partially purified insulin receptor and a random copolymer of glutamic acid and tyrosine as a substrate. Insulin-stimulated autophosphorylation of the receptor itself was not blocked by quercetin (up to 500 microM). In intact rat adipocytes, quercetin inhibited insulin-stimulating effects on glucose transport, oxidation, and its incorporation into lipids. Inhibition of lipogenesis (50%) occurred at 47 +/- 4 microM, whereas full inhibition was evident at 110 +/- 10 microM quercetin. In contrast, the effect of insulin in inhibiting lipolysis remained unaltered in quercetin-treated adipocytes. The inhibitor was devoid of general adverse cell affects. Basal activities and the ability of lipolytic agents to stimulate lipolysis were not affected. Inhibition by quercetin enabled us to evaluate which insulinomimetic agents are dependent on tyrosine phosphorylation of endogenous substrates for stimulating glucose metabolism. Quercetin blocked lipogenesis mediated by insulin, wheat germ agglutinin, and concanavalin A. The lipogenic effect of Zn2+ and Mn2+ was partially blocked, whereas that of vanadate was not affected at all.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quercetin metabolism in the lens: role in inhibition of hydrogen peroxide induced cataract

Oxidative stress is implicated in the initiation of maturity onset cataract. Quercetin, a major flavonol in the diet, inhibits lens opacification in a lens organ culture oxidative model of cataract. The aim of this research was to investigate the metabolism of quercetin in the lens and show how its metabolism affects the ability to prevent oxidation-induced opacity. The LOCH model (Free Radical Biology & Medicine 26:639; 1999) was employed, using rat lenses to investigate the effects of quercetin and metabolites on hydrogen peroxide-induced opacification. High-performance liquid chromatography analysis showed that the intact rat lens is capable of converting quercetin aglycone to 3'-O-methyl quercetin (isorhamnetin). Over a 6 h culture period no further metabolism of the 3'-O-methyl quercetin occurred. Loss of quercetin in the lens was accounted for by the increase in 3'-O-methyl quercetin. Incubation with 3,5-dinitrocatechol (10 microM), a catechol-O-methyltransferase (COMT) inhibitor, prevented the conversion of quercetin to 3'-O-methyl quercetin. The presence of both membrane-bound and soluble COMT was confirmed by immunoblotting. The results demonstrate that in the rat lens COMT methylates quercetin and that the product accumulates within the lens. Quercetin (10 microM) and 3'-O-methyl quercetin (10 microM) both inhibited hydrogen peroxide- (500 microM) induced sodium and calcium influx and lens opacification. Incubation of lenses with quercetin in the presence of COMT inhibitor revealed that the efficacy of quercetin is not dependent on its metabolism to 3'-O-methyl quercetin. The results indicate dietary quercetin and metabolites are active in inhibiting oxidative damage in the lens and thus could play a role in prevention of cataract formation.     

Two sensitivity levels of cattle oocytes to puromycin

Germinal vesicle breakdown (GVBD) in cumulus-enclosed and denuded cattle oocytes was sensitive to puromycin at concentrations at or above 50 micrograms/ml. Media supplemented with 5-25 micrograms/ml of puromycin did not significantly reduce either rate or sequence of GVBD after 8 h of culture (82-96% GVBD). In concentrations of 50, 75, and 100 micrograms/ml, GVBD occurred in 15, 4, and 2% of oocytes, respectively. However, 50 micrograms puromycin/ml did postpone the time sequence of GVBD, since all treated oocytes underwent GVBD after 20 h of culture. Oocytes arrested in the germinal vesicle (GV) stage possessed GV filled with highly condensed bivalents. The puromycin block (100 micrograms/ml) was fully reversible, and the time sequence of GVBD was two times faster than in control medium. Proteins important for GVBD were synthesized during the first 4 h of culture, and 81% of oocytes underwent GVBD when puromycin (100 micrograms/ml) was added after 4 h of preincubation in control medium. The first polar body (I PB) expulsion was more sensitive to inhibition of protein synthesis, as shown by the observation that 2.5 and 5 micrograms puromycin/ml significantly (69 and 61%) reduced the incidence of Metaphase II, and 10 micrograms/ml highly significantly (31%) reduced it. The I PB expulsion in concentrations of 25 and 37 micrograms puromycin/ml was less than 5%. The subsequent culture in puromycin (8 h) and 6-dimethylaminopurine (8 h) proved that nuclear membrane breakdown is less sensitive to inhibition of protein phosphorylation than the process of chromatin condensation.     

Regulation of M-phase progression in Chaetopterus oocytes by protein kinase C.

We have examined the presence of protein kinase C in oocytes of Chaetopterus pergamentaceus and its role in the initiation of germinal vesicle breakdown (GVBD). First, we demonstrated that the oocytes contain a phospholipid- and calcium-dependent protein kinase, protein kinase C (PKC). Since PKC is the primary intracellular receptor for phorbol esters, we tested the ability of phorbol 12,13-dibutyrate (PDBu) to induce GVBD and compared several critical events and processes involved in GVBD induced by PDBu to those induced normally (by seawater). Seawater and 100-200 nM PDBu induced chromosome condensation, spindle formation, and spindle migration over a similar time course. Both treatments induced similar alterations in the SDS-PAGE pattern of newly synthesized proteins. The synthesis of polypeptides of approximately 46 and 54 kDa increased specifically. Both treatments increased oocyte protein phosphorylation, especially of proteins of 22, 32, 46, 55, 64, and 84 kDa. Both treatments resulted in the activation of an M-phase-specific histone H1 kinase activity, which demonstrates the appearance of maturation-promoting factor. Staurosporine, a potent protein kinase C inhibitor, blocked GVBD and the activation of M-phase-specific H1 kinase, whereas HA1004, which preferentially antagonizes protein kinase A, had no effect. The results of this study demonstrate that protein kinase C can activate a wide spectrum of essential biochemical and morphological processes involved in GVBD. Further, these studies suggest that protein kinase C elicits GVBD by activating maturation-promoting factor and support the hypothesis that protein kinase C plays an essential role in oocyte maturation in this species.     

Inactivation of MAPK affects centrosome assembly, but not actin filament assembly, in mouse oocytes maturing in vitro

Mitogen-activated protein kinase (MAPK) plays a crucial role in meiotic maturation of mouse oocytes. In order to understand the mechanism by which MAPK regulates meiotic maturation, we examined the effects of the MAPK pathway inhibitor U0126 on microtubule organization, gamma-tubulin and nuclear mitotic apparatus protein (NuMA) distribution, and actin filament assembly in mouse oocytes maturing in vitro. Western blotting with antibodies that detect active, phosphorylated MAPK revealed that MAPK was inactive in fully grown germinal vesicle (GV) oocytes. Phosphorylated MAPK was first detected 3 hr after the initiation of maturation cultures, was fully active at 6 hr, and remained active until metaphase II. Treatment of GV stage oocytes with 20 microM U0126 completely blocked MAPK phosphorylation, but did not affect GV breakdown (GVBD). However, the oocytes did not progress to the Metaphase I stage, which would normally occur after 9 hr in the maturation cultures. The inhibition of MAPK resulted in abnormal spindles and abnormal distributions of gamma-tubulin and NuMA, but did not affect actin filament assembly. In oocytes treated with U0126 after GVBD, polar body extrusion was normal, but the organization of the metaphase plate and chromosome segregation were abnormal. In conclusion, the meiotic abnormalities caused by U0126, a specific inhibitor of MAPK signaling, indicate that MAPK plays an important regulatory role in microtubule and centrosome assembly, but not actin filament assembly.     

Cyclic AMP-mediated control of oocyte maturation in the catfish, Clarias batrachus (Bloch): effects of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one and phosphodiesterase inhibitors

An increase in the percentage of germinal vesicle breakdown (GVBD) with a corresponding decrease in cAMP was found in the oocytes which were incubated for 36 hr with different concentrations of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP). At its highest concentration (1 microgram/ml), 17 alpha,20 beta-DP induced 91.9 +/- 2.3% GVBD and decreased cAMP level to 0.8 +/- 0.1 pmol/oocyte from 2.9 +/- 0.2 pmol/oocyte (control). The two different known inhibitors of phosphodiesterase viz. 3-isobutyl-1-methyl-xanthine (IBMX) and theophylline inhibited GVBD in vitro and promoted the accumulation of cAMP in a dose-dependent manner irrespective of whether the oocytes were treated for a short duration (2 hr) or for a long duration (36 hr). Evaluation of time course response to 1 mM IBMX or 1 mM theophylline revealed that cAMP levels increased at all the time points when compared with their respective controls and blocked maturation. In contrast, 1 microgram/ml 17 alpha,20 beta-DP not only induced oocyte maturation but also caused an immediate decrease in cAMP within the first 2 hr (from 3.2 +/- 1.3 to 1.3 +/- 0.1 pmol/oocyte) of incubation which was maintained till the end of experiment (36 hr). Likewise, a significant inhibition of GVBD and accumulation of cAMP was recorded even in oocytes pre-stimulated with 1 microgram/ml 17 alpha,20 beta-DP for 6 hr and then treated with different concentrations of IBMX or theophylline. Taken together, these data strongly suggest that in C. batrachus a decrease of oocyte cAMP concentration is a prerequisite for the induction of oocyte maturation, and its increase is associated with the maintenance of meiotic arrest.