Inhibition Of Germinal Vesicle Breakdown Research Articles

Antioxidants reversibly inhibit the spontaneous resumption of meiosis.

We previously showed that the cell-permeant antioxidant 2(3)-tert-butyl-4-hydroxyanisole (BHA) inhibited germinal vesicle breakdown (GVBD) in oocyte-cumulus complexes (OCC) of the rat. The objective of the present studies was to assess other antioxidants and whether such inhibition was reversible. Spontaneous GVBD in OCC incubated for 2 h was significantly inhibited (P < 0.005) by nordihydroguaiaretic acid (NDGA; GVBD = 19.4%), BHA (GVBD = 25.7%), octyl gallate (OG; GVBD = 52.2%), ethoxyquin (EQ; GVBD = 58.8%), 2, 6-di-tert-butyl-hydroxymethyl phenol (TBHMP; GVBD = 59%), butylated hydroxytoluene (BHT; GVBD = 59.5%), and tert-butyl hydroperoxide (TBHP; GVBD = 60.0%). Other antioxidants that produced lower but significant (P < 0.05) inhibition of oocyte maturation included propyl gallate (PG; GVBD = 70.3%), 2,4,5-trihydroxybutrophenone (THBP; GVBD = 71.4%), and lauryl gallate (LG; GVBD = 71.4%). Antioxidants that had no effect on oocyte maturation at the same concentration (100 microM) included ascorbic acid, vitamin E, and Trolox. Inhibition of GVBD was evident for up to 8 h of incubation of OCC and denuded oocytes (DO) with BHA or NDGA and was reversed by washing. NDGA was less potent than BHA for inhibition of GVBD in DO, unlike that seen with OCC. Oocyte maturation was induced by incubation of follicles for 3 h with human chorionic gonadotropin (hCG), and this response was inhibited by BHA or NDGA. These findings support the conclusion that cell-permeant antioxidants inhibit spontaneous resumption of meiosis, which may implicate a role of oxygen radicals in oocyte maturation.

Effects of glucocorticoids on maturation of pig oocytes and their subsequent fertilizing capacity in vitro.

The aim of this study was to assess the possible role of glucocorticoids in the maturation of pig oocytes and their subsequent fertilizing capacity in vitro. Pig cumulus-enclosed oocytes collected from prepubertal gilts were cultured in Waymouth MB752/1 medium supplemented with sodium pyruvate (50 microg/ml), LH (0.5 microg/ml), FSH (0.5 microg/ml), and estradiol-17beta (1 microg/ml) in the presence or absence of cortisol or dexamethasone (DEX) for 24 h; they then were cultured without hormonal supplements in the presence or absence of cortisol or DEX for an additional 16-24 h. Treatment of cumulus-enclosed or denuded oocytes with increasing concentrations of cortisol or DEX for 48 h resulted in a dose-response inhibition of germinal vesicle breakdown (GVB). Increasing duration (12-48 h) of treatment with DEX (10 microg/ml) led to a time-dependent inhibition of GVB, which achieved statistical significance by 12 h. The addition of DEX (10 microg/ml) to maturation medium immediately after culture or at 12 h, 24 h, or 36 h after culture also decreased the percentage of oocytes with GVB. When oocytes were exposed to DEX for 48 h, the maturation rate was reduced. The degree of this reduction was dependent on DEX, and a concentration of DEX higher than 0.1 microg/ml was needed. The inhibitory effect of DEX on the maturation of oocytes was prevented by the glucocorticoid receptor antagonist RU-486. Exposure of oocytes to DEX for 40 h did not prevent sperm penetration, affect the incidence of polyspermy, or decrease the ability of oocytes to form a male pronucleus. The intracellular concentration of glutathione (GSH) in cumulus-enclosed oocytes was 4.4 mM per oocyte. Exposure of oocytes to DEX (0.01-10 microg/ml) had no effect on GSH concentration. These results demonstrate that glucocorticoids directly inhibit the meiotic but not cytoplasmic maturation of pig oocytes in vitro. This inhibitory effect is not mediated through a decrease in the level of intracellular GSH.

Inhibition of the adenylyl cyclase and activation of the phosphatidylinositol pathway in oocytes through expression of serotonin receptors does not induce oocyte maturation

The identification of the initial signaling events induced by progesterone in Xenopus oocyte maturation was approached by expressing serotonin receptors and by using pharmacological agents. Suppression of phospholipase C (PLC) activity in oocytes by U-73122 or stimulation of oocyte adenylyl cyclase (ACase) by forskolin inhibited progesterone-induced reinitiation of meiotic cell division. Microinjection of cAMP-dependent protein kinase (PKA) pseudosubstrate, PKI, into oocytes--or pretreatment of oocytes with PKA inhibitor, H-89--did not induce oocyte maturation, but both treatments potentiated the rate of progesterone-induced germinal vesicle breakdown (GVBD). In addition, reduced PKA activity by H-89 reversed the inhibition of GVBD caused by U-73122. Expression and activation of the serotonin receptor type 1a or type 2c lowered intracellular cAMP level or mobilized Ins(1,4,5)P3, respectively. These oocytes, however, did not undergo maturation upon serotonin stimulation. Co-expression of these receptors also did not trigger maturation, but it significantly accelerated the rate of GVBD induced by progesterone. From these data, we conclude that (1) changes in levels of these second messengers may well be coupled with progesterone signaling; (2) an initial decrease in cAMP and production of Ins(1,4,5)P3/DG may not be absolute requisites for progesterone-induced meiotic maturation: (3) cross-talk mechanisms between adenylyl cyclase and phosphoinositide signal transduction pathways may exist in the process of progesterone-induced reinitiation of meiotic cell cycle.

The Proteasome Is an Essential Mediator of the Activation of Pre-MPF during Starfish Oocyte Maturation

Starfish oocyte maturation was blocked by the addition of 100 μM MG115, a potent proteasome inhibitor, whereas no inhibition was observed by membrane permeable cysteine protease inhibitor, E-64-d. The inhibition by MG115 was diminished by adding at a time corresponding to the half time required for germinal vesicle breakdown. Potent inhibition of germinal vesicle breakdown was also observed by microinjection of anti-proteasome-a-subunit antibodies. The antibody-injected oocytes failed to activate pre-maturation promoting factor (pre-MPF), since the dephosphorylation of phospho-Tyr15 in cdc2 kinase was not observed even in the presence of 1-methyadenine, a maturation-inducing hormone. These results indicate that the proteasome triggers the activation of pre-MPF via the dephosphorylation of cdc2 kinase in the signal transduction pathway in response to the hormonal stimulus during starfish oocyte maturation.

Effects of cAMP forskolin and cyanoketone on invitro oocyte maturation in the catfish,Clarias batrachus

This study investigated the interactive effects of cyanoketone (CK), an inhibitor of 3β-hydroxysteroid dehydrogenase on the effects of cAMP and forskolin (FK) on oocyte maturation inClarias batrachus using an in vitro incubation technique. When the oocytes were incubated in the presence of 1 Μg/ml 17α, 20β-dihydroxy-4-pregnen-3-one[l7α, 20Β-DP, the maturation-inducing steroid (MIS) of this species] for 6h, they matured [85.3 + 1.36% germinal vesicle breakdown (GVBD)] normally after additional incubation for 20–30 h in plain medium. On the other hand, exposure to 1.0 and 8 0 mM of cAMP after MIS stimulation caused significant inhibition of GVBD but lower concentrations (0.1 and 0.5 mM) of cAMP were noninhibitory. However, when the oocytes were preincubated for 1 h with 1 μg/mI CK, a significant inhibition in the percentage of GVBD was recorded including the lower concentrations of cAMP. FK, an activator of adenylate cyclase, could significantly induce GVBD at all of its concentrations (0.1, 0.5, 1.0 and 10.0 μM) in a dose- and time-dependent manner. However, when the oocytes were exposed to 1 μg/ml CK for 1 h, prior to FK stimulation, a complete inhibition of GVBD occurred but when CK treatment was given after the FK stimulation, only a partial inhibition of maturation was observed. Taken together, these data indirectly suggest that FK induces catfish oocyte maturation probably by stimulating follicular production of Δ4 steroid ( 17α,20 β-DP)through an adenylate cyclase-c AMP-mediated pathway, a mechanism identical to the gonadotropin-induced oocyte maturation.

The in vitro effects of forskolin, IBMX and cyanoketone on meiotic maturation in Follicle enclosed catfish ( Clarias batrachus) oocytes

The effects of an adenylate cyclase activator (forskolin, FK), phosphodiesterase inhibitor (3-isobutyl-1-methyl-xanthine, IBMX) and an inhibitor of steroidogenesis (cyanoketone, CK) on germinal vesicle breakdown (GVBD) in the catfish ( Clarias batrachus) were investigated in vitro. In most of the experiments GVBD was induced by using 1 μg/ml 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), which is the maturation-inducing steroid (MIS) for this species. Adenosine 3′:5′-Cyclic monophosphate (cAMP) levels were also measured in the control, MIS-induced and/or FK- and IBMX-treated follicle-enclosed oocytes. MIS-induced GVBD was inhibited by FK (≥ 0.5 μM) or IBMX (≥1.0 mM), but oocyte exposed to 0.1 μM FK or 0.5 mM IBMX, after MIS stimulation, underwent GVBD. However, an inhibition of GVBD was recorded when the MIS-induced folliculated oocytes were preincubated with CK (1μg/ml) and subsequently treated with 0.1 μM FK. In the time course study, when the oocytes were stimulated by MIS for various time intervals and then treated with 1.0 μM FK or 1.0 mM IBMX, both the substances blocked maturation if they were added up to 12 hr after MIS. The extent of inhibition was gradually decreased and was completely removed after 30 hr of post-MIS stimulation. The stimulatory dose of 17α,20β-DP (1 μg/ml) not only induced GVBD (83.2 ± 1.50%) in vitro but also reduced oocyte cAMP level ( 65.3 ± 2.85 pmol 100 μ g protein ) significantly after 6 hr of incubation. However, FK (10.0 μM) or IBMX (1.0 mM) countered these effects and promoted the accumulation of cAMP in the oocytes; FK being more potent. On the other hand, when unstimulated full-grown but immature oocytes were cultured in vitro in the presence of different concentrations of FK, an induction of oocyte maturation was recorded in dose- and time-dependent manner. These results strongly suggest the involvement of cAMP in the regulation of catfish oocyte maturation.

Effect of gonadotropins on the ability of 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB) to inhibit germinal vesicle breakdown in bovine oocytes

The transcriptional inhibitor, 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB), prevents germinal vesicle breakdown (GVBD) in bovine oocytes only in the presence of gonadotropins. The objectives of this study were to examine the ability of gonadotropins to facilitate transcriptional inhibition of GVBD in bovine oocytes and to examine the effect of gonadotropins on transcriptional inhibition of cumulus expansion. Cumulus-oocyte complexes (COC) from 2 to 7 mm follicles were cultured in TCM-199 with 1 μg/ml estradiol, 50 μg/ml gentamicin and hormonal treatments for 20 to 24 h at 39 °C in 5% CO 2 in air. After culture, COC were assessed for degree of cumulus expansion and oocytes were then denuded, fixed and stained to determine stage of meiosis. In the presence of LH and FSH the proportion of oocytes arrested at germinal vesicle (GV) stage was significantly increased with DRB treatment (58 vs 3% GV for LH/FSH + DRB vs LH/FSH-DRB; P < 0.001). However, maximal inhibition of GVBD by treatment with DRB could also be achieved in the presence of FSH alone (60% GV for FSH + DRB). The ability of DRB to block GVBD was significantly reduced in the presence of LH alone (20 to 28% GV for LH + DRB; P < 0.05 vs FSH + DRB), and treatment with DRB did not block GVBD in the presence of hCG (6.8 to 13.3% GV for hCG + DRB; P < 0.001 vs FSH + DRB). Inhibition of cumulus expansion by treatment with DRB occurred in the presence of either FSH or LH. Based on these results, it is suggested that DRB prevents GVBD in cultured bovine COC by interfering with a transcriptional event mediated primarily by FSH.

Evidence for a nuclear factor involved in c-myc RNA degradation during axolotl oocyte maturation.

We have previously described an in vivo heterologous system which has allowed us to study the stability of different Xenopus c-myc RNA constructs injected into axolotl oocytes. In full-grown oocytes, degradation of c-myc RNA does not occur. In mature oocytes treated with progesterone, transcripts containing the coding sequence of the gene are degraded, whereas those corresponding to the 3'UTR (untranslated region) alone are stable. In order to study the role of nuclear or cytoplasmic components in this process, degradation of injected c-myc transcripts was analysed (i) after inhibition of germinal vesicle breakdown (GVBD) in progesterone treated oocytes (ii) after induced maturation of enucleated oocytes, (iii) injection of nuclear contents into immature oocytes and (iv) after direct injection into the germinal vesicle of full-grown oocytes. Our results demonstrate the role of a nuclear factor stockpiled in the germinal vesicle of full-grown oocytes and specifically involved in the degradation of c-myc transcripts containing the coding region. Further biochemical characterization of this new nuclear component should lead to a better understanding of the post-transcriptional control of c-myc expression during oogenesis and early development.

Thein vitro effect of theophylline on 17α, 20ß-dihydroxy-4-pregnen-3-one-induced germinal vesicle breakdown in the catfish,Clarias batrachus

The effects of theophylline (a phosphodiesterase inhibitor) and cAMP on 17α, 20s-dihydroxy-4-pregnen-3-one-induced germinal vesicle breakdown was investigatedin vitro in catfish (Clarias batrachus) oocytes. Folliculated oocytes incubated with 17α, 20s-dihydroxy-4-pregnen-3-one at the concentration of 1 μg/ml induced 93.2 ± 2.23% germinal vesicle breakdown. When the oocytes were prestimulated with 17α,20s-dihydroxy-4-pregnen-3-one for 6 h and then treated with different concentrations of theophylline, there was a significant drop in the frequency of germinal vesicle breakdown at the concentrations 2.0, 1.5 and 1.0 mM. However, theophylline was found to be incapable of inhibiting germinal vesicle breakdown at its lowest concentration (0.5 inM). In the time course study, significant inhibition of germinal vesicle breakdown was recorded when 1 mM theophylline was added up to 30 h of 17α,20s-dihydroxy-4-pregnen-3-one Stimulation but the inhibitory effect of theophylline gradually (time dependent manner) declined if the stimulatory time of 17α,20s-dihydroxy-4-pregnen-3-one was increased. A similar inhibition of germinal vesicle breakdown was also recorded with various concentrations of cAMP. Except 0.5 mM, all the higher concentrations of cAMP significantly inhibited 17α,20s-dihydroxy-4-pregnen-3-one induced germinal vesicle breakdown.

Effects of actinomycin d and cycloheximide on gonadotropin- or 17α, 20β-dihydroxy-4-pregnen-3-one-inducedin vitro maturation in oocytes of the catfish,Clarias batrachus

The effect of inhibitors in the oocyte maturation ofClarias batrachus, was investigatedin vitro using actinomycin D and cycloheximide. Full-grown immature oocytes incubated with salmon gonadotropin (SG-G100) and 17α, 20s-dihydroxy-4-pregnen-3-one at the concentration of 1 μg/ml induced 86.0 ± 1.2% and 91.3 ± 2.4% of germinal vesicle breakdown, respectively. When the oocytes were incubated with SG-G100 (1 μg/ml) + different concentrations of actinomycin D or cycloheximide, a significant drop in the frequency of germinal vesicle breakdown was observed. Thus, gonadotropin-induced maturation was inhibited by both transcriptional and translational inhibitors. When the oocytes were incubated with 17α, 20s-dihydroxy-4-pregnen-3-one (1μg/ml) + different concentrations of cycloheximide, a significant inhibition of germinal vesicle breakdown was recorded. However, the maturation was not inhibited when the oocytes were incubated in the presence of 17α, 20s-dihydroxy-4-pregnen-3-one (1 μg/ml) and different concentrations of actinomycin D. This suggests that mRNA synthesis is not obligatory for 17α, 20β-dihydroxy-4-pregnen-3-one induced oocyte maturation. Based on the time course experiment, it was observed that the inhibition of maturation in cycloheximide treated oocytes extends up to 12 h after which the effect becomes slowly subdued.

Inhibition of germinal vesicle breakdown in bovine oocytes by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB).

5,6-Dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) is an analog of the nucleoside adenosine that has been used to inhibit transcription in a variety of cell types. The present studies were designed to evaluate the ability of DRB to block germinal vesicle breakdown (GVBD) in bovine oocytes matured in vitro and to characterize culture conditions required for DRB-mediated arrest of meiosis. Administration of DRB (60-90 microM) at 2-3 h intervals during culture of COC blocked GVBD in approximately 70 percent of oocytes. The inhibitory effect of DRB was reversible and required the presence of cumulus cells. Treatment with DRB was associated with a 57% decrease in 3H-uridine incorporation into total COC RNA and a 90.8% decrease into mRNA but did not affect the incorporation of 3H-leucine into COC proteins. The ability of DRB to arrest meiosis was significantly compromised if supplemental gonadotropin preparations were absent from the maturation media. Gonadotropin-induced GVBD as well as cumulus cell expansion was blocked by treatment with DRB but not with adenosine. GVBD in cultured bovine COC was initially inhibited and then stimulated when supplemental gonadotropin preparations were included in the culture media. DRB treatment in the presence of gonadotropin supplementation blocked the stimulatory effect of gonadotropins on GVBD. In conclusion, DRB can be used to arrest GVBD in bovine COC in a specific and reversible manner. The data support the hypothesis that gene transcription is required for the stimulatory phase of gonadotropin-mediated GVBD in cultured bovine COC.

Bovine activin A does not affect the in vitro maturation of bovine oocytes

The effect of recombinant bovine activin A on the in vitro maturation of bovine oocytes was investigated. Culture of cumulus enclosed bovine oocytes in the presence of activin at the concentration of 100 or 500 ng/ml did not change the proportion of oocytes in which germinal vesicle breakdown had occurred at 4 and 7 h after the onset of culture. Activin had also no effect on the progression of maturation to the M II stage. The transient inhibition of germinal vesicle breakdown by 10 mM dibutyryl cyclic AMP was not affected by the addition of activin A at the onset of culture. Radiolabeling with 35S-methionine at 4 h and at 18 h after culture in the presence or absence of activin A did not show any effect of activin either on the total incorporation of radiolabel into acid precipitable material or on the protein synthesis patterns obtained after SDS-PAGE.

Inhibition of meiotic maturation in growing pig oocytes by factor(s) from cumulus cells

Total inhibition of germinal vesicle breakdown (GVBD) was observed in growing pig oocytes (internal diameter 80, 90 and 100 microns) when they were cultured in a medium conditioned by cumulus oocyte complexes (COCs) of fully grown oocytes. In denuded growing oocytes, only partial inhibition was observed. The inhibitory effect was fully reversible. The addition of heparin (300 IU/ml) could overcome the effect of the conditioned medium. Transient exposure (6 h) of oocytes to dibutyryl cyclic adenosine monophosphate (dbcAMP) (1 mg/ml) could also partly reverse the effect of factor (s) produced by cumulus cells of fully grown oocytes. Follicle-stimulating hormone (5 micrograms/ml) was able to increase the percentage of maturing oocytes. The addition of luteinizing hormone (5 micrograms/ml) had no effect on GVBD inhibition by cumulus-conditioned medium.

Defining a role for calcium in the resumption and progression of meiosis in the pig oocyte

The role of calcium (Ca++) during spontaneous meiotic maturation of pig oocytes was examined. The hypothesis that elevations of endogenously derived intracellular Ca++ are prerequisite for germinal vesicle breakdown (GVBD) and progression through meiosis was tested. In addition, investigations were carried out to determine whether GVBD and meiotic progression were dependent upon extracellular Ca++ influx. Elevation of endogenously derived Ca++ was inhibited directly by loading cells with BAPTA, a specific Ca++-chelator, or indirectly with neomycin. Extracellular Ca++ influx was prevented by culturing oocytes in Ca(++)-deficient medium, with EGTA, or in the presence of the Ca++ channel blocker verapamil. Pretreatment with BAPTA/AM and subsequent culture in the absence of added exogenous Ca++ resulted in a similar inhibition of GVBD (1 microM BAPTA/AM vs. untreated, P < 0.01). After 4 h following follicular release, oocytes were no longer sensitive to BAPTA/AM treatment. Neomycin also significantly inhibited GVBD (0.5 mM neomycin vs. untreated, P < 0.05) as well as meiotic progression past metaphase I (0.25 mM neomycin vs. untreated, P < 0.05). The incidence of GVBD was not significantly affected when oocytes were simply cultured in Ca++ deficient medium or when cultured in the presence of EGTA or verapamil. However, progression of meiosis past GVBD to metaphase II was suppressed by reducing levels of Ca++ in the culture medium (0.68 mM Ca++ vs. 1.7 mM Ca++, P < 0.05) and by treatment with verapamil (0.25 mM verapamil vs. untreated, P < 0.05). Meiotic progression was completely blocked at metaphase I in the presence of 0.85 mM EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Maturation of Xenopus laevis oocyte by progesterone requires poly(A) tail elongation of mRNA

Meiotic maturation of Xenopus laevis oocytes by progesterone requires translation of stored maternal mRNAs. We investigated the role of poly(A) tail elongation of mRNAs during this process using cordycepin, which inhibits poly(A) tail elongation of mRNAs. When oocytes were treated with the buffer containing 10 m M cordycepin for 12 h, concentration of 3′-dATP in cytosol of oocytes increased to 0.7 m M, while that of ATP remained constant at around 1.2 m M. Incorporation of [ 32P]AMP into poly(A) mRNA was inhibited almost completely by this treatment. Progesterone-induced germinal vesicle breakdown (GVBD) was also abolished. Dose dependence of inhibition of progesterone-induced GVBD on cordycepin was similar to that of [ 32P]AMP incorporation into poly(A) mRNA. However, maturation-promoting factor-induced GVBD was unaffected by treatment of oocytes with cordycepin. Furthermore, the inhibition of GVBD by cordycepin was rescued by removal of cordycepin even in the presence of actinomycin D. Therefore, we concluded that poly(A) tail elongation of mRNA is required for induction of meiotic maturation of X. laevis oocytes. In addition, progesterone induced a 2.7-fold activation of [ 32P]AMP incorporation into the poly(A) tail of mRNA after a lag period of 3 h whereas GVBD was induced after 6–8 h from the progesterone treatment. Syntheses of most of the proteins were unaffected by treatment of oocytes with progesterone or cordycepin. However, syntheses of several proteins were increased or decreased by progesterone and cordycepin treatment.

Inhibition of germinal vesicle breakdown of isolated cumulus-free mouse oocytes in culture by a calcium inhibitor, TMB-8.

Inhibition of germinal vesicle breakdown of isolated cumulus-free mouse oocytes in culture by a calcium inhibitor, TMB-8.

Inhibition of germinal vesicle breakdown by 5,6-dichlorobenzimidazole riboside in bovine oocytes matured in vitro

Inhibition of germinal vesicle breakdown by 5,6-dichlorobenzimidazole riboside in bovine oocytes matured in vitro

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.

Neomycin, an inhibitor of phosphoinositide hydrolysis, inhibits the resumption of bovine oocyte spontaneous meiotic maturation.

The possibility that the intracellular signals generated upon phosphoinositide hydrolysis are involved in regulating bovine oocyte spontaneous meiotic resumption was investigated. Oocytes were mass-harvested and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin in the presence or absence of neomycin (an inhibitor of phosphoinositide hydrolysis) or phorbol myristate acetate (an activator of protein kinase C). The role of intracellular calcium was examined by preloading with BAPTA/AM (a calcium chelator) prior to culture. Meiotic maturation was scored cytogenetically. 1) Neomycin induces an irreversible inhibition of germinal vesicle breakdown which does not exceed 60% and is apparent at concentrations of 5 mM or above. Progression of meiosis past metaphase I is inhibited at concentrations of 2.5 mM or above. The full effect of neomycin is only apparent if it is presented to the oocytes within 3 h of follicular release, although germinal vesicle breakdown is not observed until 9 h culture under control conditions. 2) PMA alone has negligible effect on germinal vesicle breakdown, but it acts synergistically with 2 mM IBMX to inhibit this process. PMA has a dual effect on the progression of meiosis past metaphase I: 1 nM PMA has a stimulatory effect while 1 microM PMA blocks the ability of oocytes to reach anaphase I or beyond. These observations are not found with a non-tumor-promoting phorbol ester. 3) Spontaneous meiotic resumption is not significantly affected in the absence of added exogenous calcium. However, oocytes preloaded with BAPTA/AM exhibit a dose-dependent inhibition of germinal vesicle breakdown, even in the presence of extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of forskolin, cAMP, and cyanoketone on steroid-induced meiotic maturation of yellow perch ( Perca flavescens) oocytes in vitro

Intact yellow perch ( Perca flavescens) follicles stimulated by 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-PG) to undergo germinal vesicle breakdown (GVBD) in vitro were incubated with several agents which have been shown to increase cellular cAMP levels. Two phosphodiestrase inhibitors, SQ20,006 and isobutyl-methyl-xanthine, blocked GVBD at 1.0 m M. At lower levels (0.5, 0.1 m M) there was a dose-response effect and SQ20,006 was more inhibitory. Forskolin at 1.0–20.0 μ M blocked steroid-induced GVBD, but levels of 0.1 μ M or less were noninhibitory. In time-course experiments, significant inhibition of GVBD was observed when SQ20,006 (1.0 m M) was added within 6 hr after steroid stimulation or forskolin (10.0 μ M) was added within 12 hr. When SQ20,006 was administered in 6-hr pulses and then removed, inhibition was observed only when the steroid was given as a 1-hr prepulse which was removed at the start of the incubation period. In this case, GVBD was blocked if the SQ20,006 pulse was given before 18 hr. At 10.0 m M, cAMP completely inhibited GVBD but was noninhibitory at lower levels. However, lower levels of cAMP (1.0, 0.5 m M) and forskolin (0.1 μ M) were inhibitory if the follicles were also incubated with 1.0,μg/ml of cyanoketone, an inhibitor of steroidogenesis. These results indicate that in vitro, increases in cAMP are inhibitory to steroid-induced meiotic maturation but may stimulate steroidogenesis in the follicle wall as well. Furthermore, in vitro steroid-stimulated maturation can be inhibited by increased cAMP for a relatively long time, following steroid treatment.