The development of competence for meiotic maturation during oogenesis in Xenopus laevis

Meiotic maturation of large, 1.2-1.4 mm in diameter, stage VI oocytes of Xenopus laevis can be induced to mature in vitro by exposure to progesterone or by microinjection of maturation-promoting factor (MPF). Small, 0.95 mm in diameter, stage IV oocytes do not respond to progesterone but do undergo germinal vesicle breakdown (GVBD) in response to microinjection of MPF. The possibility that small oocytes are nonresponsive to progesterone due to a specific defect in an event known to occur with large oocytes is investigated. Both large and small oocytes possess a plasma membrane steroid receptor (Mr = 110,000) as measured by photoaffinity labeling with [3H]R5020, but the density of receptors in small oocytes is only 20% of that in large oocytes. Adenylate cyclase activity stimulated by guanyl-5'-yl-imidodiphosphate is equally inhibited by steroid (50%) in plasma membranes from both large and small oocytes with an apparent IC50 of 2 X 10(-7) M progesterone. Microinjection of the heat-stable inhibitor protein of cAMP-dependent protein kinase induces GVBD in large but not in small oocytes. These results indicate that the nonresponsiveness of small, stage IV oocytes to progesterone is due to a deficiency in an event(s) subsequent to cAMP fluctuations but prior to MPF action.     

The maturation response of stage IV, V, and VI Xenopus oocytes to progesterone stimulation in vitro

Full-grown Xenopus oocytes, Stage VI (1200-1300 microns), undergo meiotic maturation when exposed to progesterone. Smaller stage IV (800 microns) and stage V (1000 microns) oocytes remain in prophase arrest when exposed to this steroid. The larger stage VI oocytes undergo an intracellular alkalization from 7.2 to 7.6, a six- to eightfold increase in the phosphorylation of the 40 S ribosomal protein S-6, and a two- to threefold increase in total protein synthesis when exposed to progesterone. It was found that 800- to 1000-microns oocytes do not undergo these physiological changes when exposed to progesterone. This lack of response could explain the failure of small oocytes to undergo germinal vesicle breakdown (GVBD). However, when stage IV and V oocytes were artificially alkalized to a pHi of 7.6 by the weak bases, trimethylamine, procaine, or methylamine, S-6 phosphorylation was stimulated four- to sixfold and protein synthesis was stimulated two- to threefold, but they still did not undergo GVBD. Stage IV and V oocytes are able to amplify MPF injected into their cytoplasm and undergo GVBD. Thus, 800- to 1000-microns oocytes appear to contain a store of inactive MPF in their cytoplasm. It seems that an additional physiological parameter(s), that is unique to steroid-treated stage VI oocytes, is responsible for activating this MPF which induces GVBD.     

Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.     

Acid Release during Activation of Barnea candida (Mollusca, Pelecypoda) Oocytes

Barnea caridida oocytes release acid (1.35 pmole H⁺/oocyte) upon fertilization. After artificial activation by an excess of KCl, germinal vesicle breakdown (GVBD) occurs normally and a quite similar, but not identical, acid release is recorded (1.10 pmole H⁺/oocyte). KCl activation of Barnea oocytes is completely inhibited in 100 mM sodium-acetate sea water at pH 6.5 and fertilization does not result in activation when the oocytes are transferred after one minute into 100 mM sodium-acetate sea water at pH 6.3. When D–600, a calcium transmembrane fluxes inhibitor, is added 20 seconds after fertilization, GVBD is inhibited but a normal acid release is recorded. The presence of at least 10 mM sodium ions in the external medium is required for 100% activation of these oocytes by an excess of KCl. These results suggest that while an intracellular pH increase may be a requisite for GVBD, this can not be a sufficient condition to trigger it unless a calcium influx is allowed to occur. Moreover, the acid release does not result from a Ca⁺⁺-H⁺ exchange transport but appears more likely to be due to a Na⁺-H^* exchange as it has been demontrated in sea urchin eggs.     

The role of the maturation-promoting factor in controlling protein synthesis in Xenopus oocytes

Oocyte cytosol, containing maturation-promoting factor activity, induces a twofold increase in the rate of protein synthesis as well as inducing germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. In the current study, it is shown that the cytosol activity responsible for inducing the increase in protein synthesis can be separated from the activity that induces GVBD in recipient oocytes.     

Effect of ouabain on the meiotic maturation of stage IV-V Xenopus laevis oocytes

Full-grown stage VI Xenopus laevis oocytes (1,200 to 1,300 micron) respond to progesterone stimulation by undergoing a series of physiological and morphological changes that are referred to as meiotic maturation. Oocytes in earlier stages of oogenesis (I through V) do not undergo these changes and remain in prophase arrest when exposed to this steroid. We have found that oocytes ranging from 850 micron (stage IV) to 1,000 micron (stage V) are capable of responding to progesterone under the appropriate conditions. Oocytes greater than or equal to 850 micron in diameter underwent germinal vesicle breakdown (GVBD) after 10-12 hr of exposure to progesterone when ouabain was added to the medium at a concentration greater than 2.5 X 10(-6) M. Under this culture condition, progesterone was now able to induce a 0.3- to 0.4-unit increase in the intracellular pH of stage IV-V oocytes, a 4- to 5-fold increase in 40s ribosomal protein S-6 phosphorylation, and a 2.3-fold increase in their rate of protein synthesis. All of these physiological changes are characteristic of full-grown stage VI oocytes undergoing meiotic maturation. In addition, we have found that oocytes greater than or equal to 750 micron are capable of amplifying maturation promoting factor (MPF) in their cytoplasm leading to GVBD. Therefore, stage IV-V Xenopus oocytes have the potential for undergoing meiotic maturation, but they are blocked at a point in prophase that appears to be alleviated by the combination of progesterone and ouabain.     

MPF is activated in growing immature Xenopus oocytes in the absence of detectable tyrosine dephosphorylation of P34cdc2.

Tyrosine-phosphorylated p34cdc2 and cyclin B2 are present and physically associated in small growing stage IV oocytes (800 microns in diameter) of Xenopus laevis. Microinjection of M-phase promoting factor (MPF) into stage IV oocytes induces germinal vesicle breakdown and the activation of the kinase activity of the p34cdc2/cyclin B2 complex measured on p13suc1 beads. During the in vivo activation of MPF in stage IV oocytes, p34cdc2 tyrosine dephosphorylation is not detectable, in contrast to stage VI oocytes. Addition of cycloheximide in MPF-injected stage IV oocytes induces neither the inhibition of histone H1 kinase activity nor the cyclin B2 degradation. Therefore, the activation mechanism of histone H1 kinase in stage IV oocytes does not require detectable tyrosine dephosphorylation of p34cdc2. It is suggested rather that the tyrosine phosphorylation of p34cdc2 plays a role in inhibiting cyclin B2 degradation.     

One Millimeter large Oocytes as a Tool to Study Hormonal Control of Meiotic Maturation in Starfish: Role of the Nucleus in Hormone-stimulated Phosphorylation of Cytoplasmic Proteins

Single nuclei (germinal vesicles) manually isolated from large oocytes of the starfish Echinaster sepositus , as well as the complementary anucleated oocytes, were used to investigate the early changes of protein phosphorylation which occur from 1-MeAde addition to germinal vesicle breakdown (GVBD). Stimulation of protein phosphorylation was already evident in the nucleus shortly after 1-MeAde addition (18 min, thus about 0.40x the time required for GVBD), although it began first in the cytoplasm. No translocation of phosphoprotein across the nuclear envelope was detected before GVBD. Presence of the nucleus is not required for the hormone to stimulate protein phosphorylation in the remaining part of the oocytetin:fact the patterns of protein phosphorylation in enucleated oocytes were found to be identical, whether enucleation was performed after or before hormonal treatment. Cytoplasm taken at the time of GVBD from maturing Echinaster oocytes induces meiotic maturation when transferred in stage VI immature oocytes of the amphibian Xenopus laevis.     

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.     

PROPIONATE AND CYCLOHEXIMIDE REVERSIBLY BLOCK PROGESTERONE INDUCED CALCIUM SURGE IN AMBYSTOMA MEXICANUM OOCYTES

The protoprotein aequorin was used in order to monitor Ca²⁺ transients in conditions where progesterone induced maturation was reversibly inhibited. Propionate but not isethionate Cl-free medium impaired both meiosis reinitiation and the Ca²⁺ transient, unless oocytes were returned to normal Cl-containing medium. Similar results were obtained with the protein synthesis inhibitor cycloheximide. In both cases, the incidence of germinal vesicle breakdown (GVBD) and the time schedule relating it to the Ca²⁺ surge appeared not very different from that found from control oocytes. The evidence suggests that both treatments act on the initial step by which progesterone triggers the intracellular Ca²⁺ release needed for maturation promoting factor (MPF) elaboration. No definitive conclusion can be reached however from these experiments concerning the need for protein synthesis during meiosis reinitiation.     

Nature of the 1-Methyladenine-Requiring Phase in Maturation of Starfish Oocytes

Reinitiation of meiosis in starfish oocytes requires the continuous presence of 1-methyladenine (1-MeAde) in the surrounding medium for a definite period. The length of the 'hormone-dependent phase' (HDP) in Asterina pectinifera , which was defined as the time necessary for induction of 50% germinal vesicle breakdown (GVBD), was found to be about 11 min at 17°C, and 8 min at 20°C. Repeated treatments for shorter periods with 1-MeAde revealed that the action of this agent was cumulative, and that stable intermediate states between the unstimulated and fully stimulated levels existed during the HDP. Measurement of the stiffness of oocytes also demonstrated this stable intermediate state. Thus, there may be a factor(s) in the cytoplasm that accumulates continuously during the HDP and triggers GVBD when it reaches a critical level(s). When dithiothreitol (DTT) was used as an artificial maturation-inducing agent, the intermediate state was far less stable, suggesting a difference in the modes of action of 1-MeAde and DTT. Isotonic CaCl₂, the Ca²⁺ ionophore (A 23187) and methylxanthines, which are known to cause increase in intracellular Ca²⁺, had additive effects with 1-MeAde. These results suggest that part of the action of 1-MeAde is to release Ca²⁺ in the oocyte cytoplasm.     

The effect of insulin on intracellular ph and ribosomal protein S6 phosphorylation in oocytes of Xenopus laevis

Both insulin and progesterone are capable of stimulating germinal vesicle breakdown (GVBD) of large, Stage VI oocytes of Xenopus laevis. Numerous studies have shown an increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation prior to GVBD in oocytes treated with progesterone. In this study the effect of insulin and progesterone on pHi and S6 phosphorylation was compared. Both hormones increased pHi and S6 phosphorylation to similar levels and the time course of pHi change was the same for both hormones. Half-maximal effects of insulin were observed at 7 X 10(-8) M concentrations. In the presence of 1 nM cholera toxin, the ability of progesterone to induce these two responses was inhibited while the action of insulin was unaffected. However, GVBD induced by either hormone was blocked by cholera toxin. In small, Stage IV oocytes that do not undergo GVBD in response to either progesterone or insulin, a partial increase in pHi without S6 phosphorylation occurred in response to progesterone but both events occurred in response to insulin. These results suggest that the inability of Stage IV oocytes to undergo GVBD in response to hormone is not due to a failure to increase pHi or phosphorylate S6. The results in this paper also indicate that these events are regulated differently by insulin and progesterone in Xenopus oocytes.     

Change in Intracellular Calcium Ions upon Maturation in Starfish Oocytes

A transient increase in intracellular Ca²⁺ upon maturation in starfish oocyte was revealed by light emission of aequorin microinjected into the cell. One minute application of 1-methyladenine (1-MeAde) to a limited area of the oocyte surface was sufficient to induce the Ca²⁺ transient over the entire cell though it did not induce the germinal vesicle breakdown (GVBD). Ten minutes application of 1-MeAde induced a similar Ca²⁺ transient followed by GVBD. Even when the transient increase of Ca²⁺ was inhibited by injecting EGTA into the oocyte, 1-MeAde treatment for a long period induced GVBD. These facts indicate that the Ca²⁺ increase is neither necessary nor sufficient for maturation of the starfish oocyte.
When the oocyte, which had been treated with 1-MeAde for 1 min at a limited area around the animal pole, was treated again with 1-MeAde for 10 min starting about 15 min after the first treatment, a Ca²⁺ transient similar to the first one was induced and was followed by GVBD. By contrast, in the oocyte treated with 1-MeAde at an area around the vegetal pole, neither Ca²⁺ transient nor GVBD was induced by the second treatment with 1-MeAde. These results indicate a difference in responsiveness to the hormone between the animal hemisphere and the vegetal hemisphere of the oocyte.     

5S RNA AND TRANSFER RNA SYNTHESIS IN GERMINAL VESICLE OOCYTES AND DURING HORMONALLY-INDUCED MEIOTIC MATURATION OF STARFISH OOCYTES

The incorporation of ³H-guanosine as ³H-GMP into 5S RNA and into transfer RNA (tRNA) was examined in isolated large germinal vesicle oocytes, in isolated mature ootids and during and subsequent to hormonally (l-methyladenine)-induced meiotic maturation in the starfish, Asterias forbesii .Purified soluble RNA ¹ preparations at each stage were fractionated by electrophoresis on 10% polyacrylamide gels, while high molecular weight RNAs were resolved by subjecting total RNA samples to electrophoresis on 2.4% acrylamide+0.5% agarose gels. The results showed that large germinal vesicle oocytes, containing a single compact nucleolus, synthesize 5S RNA and tRNA as well as the previously-reported (1, 23-26) nucleolar rRNAs. In contrast, during and subsequent to hormonally-induced meiotic maturation, after germinal vesicle braekdown and nucleolar dissolution, the synthesis of 5S RNA and tRNA continues in the absence of detectable high molecular weight rRNA synthesis.     

On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis

We have measured the levels of cyclin mRNAs and polypeptides during oogenesis, progesterone-induced oocyte maturation, and immediately after egg activation in the frog, Xenopus laevis. The mRNA for each cyclin is present at a constant level of approximately 5 x 10(7) molecules per oocyte from the earliest stages of oogenesis until after fertilization. The levels of polypeptides show more complex patterns of accumulation. The B-type cyclins are first detectable in stage IV and V oocytes. Cyclin B2 polypeptide is present at approximately 2 x 10(9) molecules (150 pg) per oocyte by stage VI. The amount increases after progesterone treatment, but returns to its previous level after GVBD and undergoes no further change until it is destroyed at fertilization. Cyclin B1 is present at 4 x 10(8) molecules per oocyte in stage VI oocytes, and rises steadily during maturation, ultimately reaching similar levels to cyclin B2 in unfertilized eggs. Unlike the B-type cyclins, cyclin A is barely detectable in stage VI oocytes, and only starts to be made in significant amounts after oocytes are exposed to progesterone. A portion of all the cyclins are destroyed after germinal vesicle breakdown (GVBD), and cyclins B1 and B2 also experience posttranslational modifications during oocyte maturation. Progesterone strongly stimulates both cyclin and p34cdc2 synthesis in these oocytes, but whereas cyclin synthesis continues in eggs and after fertilization, synthesis of p34cdc2 declines strongly after GVBD. The significance of these results is discussed in terms of the activation and inactivation of maturation-promoting factor.     

An increase of intracellular free Ca2+ is essential for spontaneous meiotic resumption by mouse oocytes.

The involvement of calcium ions in the mechanism of meiotic resumption has been studied in mouse oocytes made resistant to the lethal effects of calcium-free medium (CFM) by zona pellucida removal (De Felici et al., '89). We show here that such oocytes undergo meiotic resumption in CFM (as evaluated by germinal vesicle breakdown, GVBD) at a rate comparable to that shown by oocytes cultured in medium containing 1.7 mM Ca2+. The addition to CFM of 50 u M Quin2/AM (a membrane permeable, high affinity Ca2+ chelator) totally prevents GVBD, while purported antagonists of Ca2+ release from intracellular stores, such as 150 uM 8-(N,N-diethylamino)octyl-3-4-5 trimethoxybenzoate (TMB-8) or 300 uM chlortetracycline, only cause a slight meiotic delay. On the other hand, if the oocytes are pre-incubated for 30 min in CFM supplemented with 100 uM TBM-8 plus 0.2 mM dibutyryl-cyclic AMP (dbcAMP, a reversible inhibitor of GVBD), and then cultured in the same medium, without dbcAMP, a sustained inhibition of meiotic maturation is obtained. Our observations suggest that an increase in intracellular free Ca2+ is essential for meiotic resumption by mouse oocytes; in the experimental absence of external Ca2+, release of the cation from internal stores is sufficient to allow meiotic resumption.     

Short time priming of pig cumulus-oocyte complexes with FSH and forskolin in the presence of hypoxanthine stimulates cumulus cells to secrete a meiosis-activating substance

This study evaluated the effect of forskolin and FSH on pig oocyte maturation when cultured in a maturation inhibiting system. Ovaries from prepubertal gilts were collected at a local slaughterhouse. Oocytes were cultured in a hypoxanthine (HX 4 mM) containing M 199 for 24 or 40 h with or without forskolin and FSH treatment. After the culture, we examined germinal vesicle breakdown (GVBD) and polar body (PB) formation. Two experiments were designed. (1) Cumulus enclosed oocytes (CEO) were cultured for 24 or 40 h with or without different doses of forskolin and FSH. (2) CEO were primed by forskolin and FSH for different times and then transferred into an HX-medium for a further culture. The total culture period was 24 h. The results revealed that 4 mM HX markedly prevented pig CEO from undergoing GVBD. After 24 and 40 h culture, FSH (50-200 U/L) stimulated oocytes to resume meiosis by overcoming the inhibition of HX. Both GVBD and PB formation were increased (P < 0.002 and 0.01 respectively) after 40 h exposed to FSH. Forskolin showed a biphasic effect on CEO maturation. Within 24 h forskolin, in combination with HX, inhibited oocytes maturation. The GVBD percentage was significantly decreased compared to HX alone group (2% to 20%, P < 0.01), whereas no inhibition was observed after 40 h of culture. The second experiment showed that forskolin (3 microM) and FSH (100 U/L) priming CEO could time-dependently induce oocyte maturation by overriding the inhibition of HX. After 30 and 60 min priming by FSH or forskolin, the GVBD and PB percentage was significantly increased (P < 0.002 and 0.01 respectively). No difference of GVBD percentage was observed between FSH short time priming group and FSH long time presentation group. In conclusion, we found that forskolin and FSH in vitro can stimulate pig cumulus cells to secrete a meiosis-activating substance which induces the oocyte to overcome the inhibition of hypoxanthine and undergo GVBD.     

A rapid and sensitive ion chromatographic technique for the determination of sulfate and sulfate reduction rates in freshwater lake sediments

Abstract Newly developed low capacity columns were used in suppressed ion chromatography for rapid and highly reproducible determination of SO₄²⁻ in porewater samples from freshwater sediments without preconcentration of samples. With a 50 μl injection the detection limit for SO₄²⁻ was ca. 50 pmol (= 1 μ M) with a precision of 1–3% at the 10–200 μM level and <1% at concentrations above 200 μM. SO₄²⁻ could be measured in 4–5 min with the routinely used eluent (3.0 mM NaHCO₃/0.8 mM Na₂CO₃). When the strength of the eluent was increased to 3.0 mM NaHCO₃/2.0 mM Na₂CO₃, sulfate analysis was possible in less than 3 min, provided that samples were nitrate-free. Under these conditions S₂O₃²⁻ could also be sensitively determined in about 6 min. Examples of application of the method are given for measurements of sulfate reduction rates in freshwater sediment samples from Lake Constance.     

Induction of M-phase entry of prophase-blocked mouse oocytes through microinjection of okadaic acid, a specific phosphatase inhibitor.

We report that a specific inhibitor of types 1 and 2A phosphatases, okadaic acid (OA), induces germinal vesicle break down (GVBD) and chromosome condensation when microinjected into denuded mouse oocytes maintained in prophase block by analogs of cAMP, inhibitors of phosphodiesterase, or a tumor-promoting phorbol ester. GVBD and chromosome condensation are also observed when incompetent oocytes are similarly injected with OA, this effect being dependent on the oocyte diameter. Marked changes in cell shape, cytoskeletal organization, and chromosome condensation with abnormal or abortive spindle formation are associated with such injections. The polar body is not formed. These results led to the conclusions that in mouse oocytes, OA acts distal to both the cAMP-modulated pathway involved in meiotic arrest and the inhibitory action exerted by tumor-promoting phorbol esters.     

Time sequence of germinal vesicle breakdown in pig oocytes after cycloheximide and p-aminobenzamidine block

All porcine oocytes cultured 20 hr in medium with 10 μg/ml cycloheximide rested in the germinal vesicle (GV) stage but with the highly condensed bivalents in nucleoplasm. When these oocytes were washed and cultured in the control medium for 2, 4, and 6 hr, germinal vesicle breakdown (GVBD) was completed in 0, 86, and 100% of them, respectively. When similarly inhibited oocytes cultured successively only 2.5 hr in the control medium were given again in cycloheximide enriched medium (3.5 hr), nearly all of them reached late diakinesis stage again. It means that oocytes cultured for 20 hr and washed free of this inhibitor of protein synthesis completed GVBD rapidly (4 hr) and protein synthesis crucial for nuclear membrane disintegration occurred already during the first 2 hr after washing of inhibitor. All oocytes cultured for 20 hr in medium with 1 mM p-aminobenzamidine rested in GV with chromatin around the compact nucleolus. The successive culture in cycloheximide (20 hr) and p-aminobenzamidine (10 hr) prevented GVBD in all oocytes, too. In contrast, when the oocytes washed after cycloheximide block (20 hr) were cultured in p-aminobenzamidine enriched medium 2 and 3 hr and again for 6 hr in cycloheximide medium, the nuclear membrane dissolved in 62 and 68% of oocytes, respectively. These data suggest that inhibition of protein synthesis in pig oocytes does not prevent the high condensation of bivalents in GV. However, nuclear membrane breakdown requires the successive protein synthesis and proteolysis.