Protein synthesis and phosphorylation patterns of bovine oocytes maturing in vivo

To investigate protein synthesis and phosphorylation during bovine oocyte maturation in vivo, oocytes were collected at consecutive times after the preovulatory luteinizing hormone (LH) peak. Therefore, heifers treated for superovulation were ovariectomized between 3 and 20 h after the maximum of the LH peak. Subsequently, cumulus-enclosed oocytes, selected from nonatretic follicles greater than 10 mm, were radiolabeled with 35S-methionine or 32P-orthophosphate for 3 h and individually prepared for gel electrophoresis. Changes in the protein synthesis patterns were observed coinciding with germinal vesicle breakdown (GVBD). No changes were detected during the ensuing maturation period or coinciding with the extrusion of the first polar body. In addition, the protein phosphorylation patterns exhibited striking differences around GVBD. In particular, a phosphoprotein band of 19 kDa and the two heavily phosphorylated proteins with apparent molecular weights between 50 and 60 kDa were present in patterns of oocytes in the germinal vesicle stage. The results are discussed in relation to previous data obtained during maturation in vitro.     

Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.     

Protein phosphorylation patterns during in vitro maturation of the goat oocyte

Protein phosphorylation patterns were studied by radiolabelling goat cumulus oocyte complexes with [³²P]orthophosphate for various periods of time. The radiolabelled denuded oocytes were assessed for nuclear status and were used individually for gel electrophoresis. This study demonstrated that specific changes in protein phosphorylations were programmed during goat oocyte maturation. One of the most prominent changes was a general increase in the phosphorylation rate at germinal vesicle breakdown (GVBD). From 8 hr of culture, dominant phosphoprotein bands with apparent molecular weights of 27, 31, 40, and 50 kD were observed; they remained at this level until the metaphase II stage. In the molecular weight range of 65–80 kD, the protein phosphorylation pattern exhibited characteristic differences, with a complex series of phosphoproteins appearing and disappearing, during maturation. Addition of 6-dimethylaminopurine (6-DMAP) at the onset of culture blocked the maturation process after GVBD and induced a dramatic condensation of chromatin. When added at different times after GVBD, 6-DMAP invariably induced chromosome condensation. This inhibition was partly reversible; i.e., after removal of the drug, oocytes were able to progress only until metaphase l. © 1993 Wiley-Liss, Inc.     

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.     

Activation of p34(cdc2) kinase around the meiotic resumption in bovine oocytes cultured in vitro

The p34(cdc2) kinase has been identified as a protein factor that is a regulator of meiotic maturation in mammalian oocytes. To investigate the regulatory function of the meiotic resumption in bovine oocytes cultured in vitro, the changes in the phosphorylation states of p34(cdc2) kinase and the histone H1 kinase activity were examined around germinal vesicle breakdown (GVBD). All bovine oocytes just after isolation from their follicles were arrested at the germinal vesicle (GV) stage, and these extracts exhibited two (upper and lower) bands of p34(cdc2) kinase on SDS-PAGE followed by immunoblotting with an antibody against C-terminal peptide of p34(cdc2). When these oocytes were cultured for 24 h in a medium supplemented with 100 microg/ml genistein, tyrosine phosphorylation inhibitor, GVBD was induced in 85% of oocytes, indicating that the upper band of p34(cdc2) kinase in bovine oocytes at the GV stage was already fully phosphorylated tyrosine residue prior to culture. Another (middle) band of p34(cdc2) kinase between the upper and lower bands appeared in the extracts of the oocytes cultured for 4 h, and significant activation of the histone H1 kinase was found in these oocytes (67 +/- 18 fmol/h/oocyte) as compared to that in oocytes cultured for 0 h (46 +/- 11 fmol/h/oocyte). The staining intensity of the middle band and the activity of the histone H1 kinase were further increased after the initiation of GVBD at 6 h of culture, but the quantitative changes of upper and lower bands were not detected throughout the 12 h of culture. Thus, it is concluded that the dephosphorylation of p34(cdc2) kinase followed by activation of the histone H1 kinase after the onset of culture plays a key role in the resumption of meiosis in bovine oocytes.     

Protein phosphorylation in meiotically competent and incompetent mouse oocytes

Specific changes in the two-dimensional gel electrophoretic pattern of mouse oocyte phosphoproteins precede germinal vesicle breakdown (GVBD). We report that changes in the relative abundance of phosphoamino acids occurred prior to GVBD. We also report data that further strengthen the close association of the changes in phosphoprotein patterns with resumption of meiosis. The calmodulin antagonist W7, which transiently inhibits GVBD, inhibited partially at least two of the maturation-associated phosphoprotein changes, the dephosphorylation of a 60,000 Mr phosphoprotein and the phosphorylation of a 36,000 Mr protein. In oocytes from juvenile mice that were incompetent to resume meiosis, neither these changes nor the phosphorylation of proteins of Mr 24,000 and 28,000 occurred; all these changes occurred, however, in oocytes from juvenile mice that were competent to resume meiosis. The microinjection of the heat-stable inhibitor of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKI), which induces GVBD in fully grown oocytes, did not induce GVBD in meiotically incompetent oocytes. Microinjected PKI did not induce the increased protein phosphorylations associated with maturation, but it did induce the dephosphorylation of the 60,000 Mr phosphoprotein. These results provide molecular markers for commitment to resume meiosis in GV-intact oocytes and indicate a potential basis for meiotic incompetence.     

Involvement of cumulus cells stimulated by FSH in chromatin condensation and the activation of maturation-promoting factor in bovine oocytes

The effects of FSH-stimulated cumulus cells on the regulatory mechanisms of chromatin condensation and maturation-promoting factor (MPF) activation around the time of germinal vesicle breakdown (GVBD) in bovine oocytes were examined. Chromatin condensation occurred in oocytes arrested at the germinal vesicle (GV) stage by protein synthesis inhibitor, cycloheximide, but this condensation was blocked by FSH-stimulated cumulus cells. However, treatment with cyclic AMP (cAMP)-dependent protein kinase inhibitor, H-8, dramatically increased the proportion of oocytes possessing GVs with condensed bivalents. Under the condition of inhibited protein synthesis, the phosphorylation form of p34cdc2 kinase was not changed due to chromatin condensation, although the activity of histone H1 kinase was significantly increased compared with that of oocytes possessing GVs with filamentous bivalents. The cycloheximide-dependent GVBD block was overcome by okadaic acid (OA) in 48 and 13% of the oocytes in the absence and presence of FSH, respectively. An initial 6-h culture period critical for protein synthesis was necessary for OA to counteract the inhibitory effect exerted by cycloheximide on the induction of GVBD and activation of histone H1 kinase in the absence of FSH, whereas this first culture period was prolonged for 2 h in the presence of FSH. Furthermore, even in FSH-stimulated oocytes, H-8 facilitated an OA-counteracted overcome of the cycloheximide-dependent GVBD block after 2 h of initial culture for protein synthesis. From these results, it is concluded that cAMP-dependent protein kinase activity regulated by cumulus cells following FSH-stimulation requests plays a role in the complex mechanism of chromatin condensation and MPF activation leading to meiotic resumption in bovine oocytes.     

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.     

Changes in protein phosphorylation accompanying maturation of Xenopus laevis oocytes

Protein phosphorylation has been measured after injection of [³²P]phosphate into oocytes of Xenopus laevis undergoing progesterone-induced meiotic maturation. As oocytes mature, there is a burst of nonyolk protein phosphorylation several hours after progesterone exposure and shortly before germinal vesicle breakdown (GVBD). This burst is not due to changes in the specific activity of the phosphate or ATP pool. Enucleated oocytes exposed to progesterone also experience the burst, indicating the cytoplasmic location of phosphoprotein formation. When an oocyte receives an injection of cytoplasm containing the maturation-promoting factor (MPF), a burst of protein phosphorylation occurs immediately, and GVBD occurs shortly thereafter, even in the presence of cycloheximide. Under a variety of conditions promoting or blocking maturation, oocytes which undergo GVBD are the only ones to have experienced the phosphorylation burst. The results suggest that the protein phosphorylation burst is a necessary step in the mechanism by which MPF promotes GVBD.     

Effects of cycloheximide on chromatin condensations and germinal vesicle breakdown (GVBD) of cumulus-enclosed and denuded oocytes in cattle

To determine if newly synthesized protein is imperative for the resumption of meiosis in bovine follicular oocytes collected from small antral follicles, cumulus-enclosed and denuded oocytes were cultured in TCM-199 both with and without various concentrations of the protein synthesis inhibitor, cycloheximide. After 11 h of culture in inhibitor-free medium, all oocytes had undergone germinal vesicle breakdown (GVBD). However, when concentrations of more than 1.0 mug/ml cycloheximide were added to the medium, the meiotic resumption of bovine oocytes was completely blocked. This inhibitory effect of cycloheximide was fully reversible after removal of the inhibitor from maturation media. Germinal vesicle breakdown following removal of cycloheximide occurred twice as fast as in the control medium. Nevertheless, when oocytes were arrested at the germinal vesicle (GV) stage by cycloheximide, a significantly higher proportion of chromatin condensation (40 to 57%) was observed in denuded oocytes than in cumulus-enclosed oocytes (11 to 22%). Thus the cycloheximide treatment could not prevent the chromatin condensation in only denuded oocytes. We conclude that protein synthesis is a prerequisite for GVBD in bovine follicular oocytes and that cumulus cells are responsible for the complementary regulation of the chromatin condensation at the GV stage, regardless of protein synthesis in the oocytes.     

Stage-specific changes in protein phosphorylation accompanying meiotic maturation of mouse oocytes and fertilization of mouse eggs

Characteristic changes in the patterns of protein phosphorylation occur during meiotic maturation of mouse oocytes from the time subsequent to germinal vesicle breakdown, through metaphase II, and following fertilization. These changes occur during both in vitro or in vivo maturation or fertilization. Three major classes of changes in total phosphoprotein synthesis are observed. In the first class, protein phosphorylations increase from the germinal vesicle stage until just after germinal vesicle breakdown and then decrease during progression to metaphase II and after fertilization. The second class is characterized by decreases in protein phosphorylation during maturation with subsequent increases in phosphorylation of these proteins after fertilization. The third class is characterized by protein phosphorylations that remain relatively constant during maturation but increase after fertilization; phosphotyrosine phosphoproteins comprise the major species. The radiolabeled protein and phosphoprotein composition of isolated germinal vesicles was also examined, and a phosphoprotein of Mr 29,000 is found exclusively associated with the germinal vesicle. Since we have shown previously that 12-O-tetradecanoyl phorbol 13-acetate inhibits fertilization (Y.Endo, R.M. Schultz, and G.S. Kopf, submitted), we examined the effects of this compound on the phosphoprotein patterns of metaphase II eggs. 12-O-Tetradecanoyl phorbol 13-acetate treatment stimulates the phosphorylation of a specific phosphoprotein of Mr 80,000.     

Biochemical studies on goat oocytes: Timing of nuclear progression, effect of protein inhibitor and pattern of polypeptide synthesis during in vitro maturation

Temporal progression of nuclear events of goat oocytes matured in vitro was studied by adding a specific inhibitor to the culture medium at different time points, to investigate protein synthesis requirements and its pattern during in vitro maturation. Goat cumulus-oocyte complexes (COCs) were matured in vitro in TCM 199, fixed at different time intervals and stained with orcein to assess nuclear changes. The germinal vesicle (GV) stage was found to be present at 0 h, chromosomal condensation stage was observed at 8 h, metaphase I at 12 to 14 h, and metaphase II was begun after 16 h of maturation and was nearly completed at 24 h. Protein synthesis inhibitor, cycloheximide, blocked oocyte maturation at germinal vesicle breakdown(GVBD), if added to the maturation medium between 0 to 4 h, suggesting that protein synthesis is required for GVBD. The transition from metaphase I to metaphase II was also protein synthesis-dependent, as observed when cycloheximide was used between 8 to 10 h of culture. When cycloheximide was added from 12 h of culture onwards, nuclear progression to metaphase II was progressively restored, but many chromosomal abnormalities were noted. Changes in the protein synthesis pattern were studied by radiolabeling of oocytes with [(35)S]-methionine at 0, 7, 12 and 24 h of culture, corresponding with GV, GVBD, metaphase I and metaphase II stages. A polypeptide of 28.1 KDa appeared as a major band at the GV stage, and its size decreased greatly and disappeared after the GVBD stage. Three new polypeptides (35, 36.5 and 39 KDa) appeared at GVBD and were detectable at metaphase II. In conclusion, the synthesis of proteins is required for the maintenance and transition of goat oocytes from GV to metaphase II during in vitro maturation.     

Requirement for protein synthesis during the onset of meiosis in bovine oocytes and its involvement in the autocatalytic amplification of maturation-promoting factor

The present study was carried out using the method of electrofusion, or treatment with okadaic acid (OA), to determine whether protein synthesis at the onset of culture was required for the meiotic resumption of bovine follicular oocytes. Germinal vesicle breakdown (GVBD) occurred in bovine oocytes at 6 hr after separation from their follicles in vitro. Following this, immature germinal vesicle (GV) oocytes, preincubated for 0,2,4, and 6 hr, were fused to mature oocytes. When immature oocytes, preincubated for 0 hr, were fused to mature oocytes and then cultured for 3 hr in basic medium, GVBD was observed in all fused cells, whereas in the case of cultivation in medium supplemented with the protein synthesis inhibitor (25 μg/ml cycloheximide; CX), 39% of the fused cells possessed an intact GV within their cytoplasm. In immature oocytes preincubated for 4 or 6 hr, however, this proportion was significantly reduced to 7% and 4%, respectively, without protein synthesis after fusion. In addition, the CX-dependent block of GVBD could be overcome in only 13% of bovine follicular oocytes by the addition of 2 μM OA, although 51% of oocytes which synthesized the protein during the first 6 hr of culture induced GVBD in subsequent culture with CX plus OA. Thus, we conclude that the initiation of GVBD in bovine oocytes requires protein synthesized at the onset of meiosis, which is related to the autocatalytic amplification of the maturation-promoting factor. © 1995 Wiley-Liss, Inc.     

Two-dimensional polyacrylamide gel analysis of changes in protein phosphorylation during maturation of Xenopus oocytes

A three- to five-fold increase in non-cAMP-dependent protein phosphorylation has previously been found to occur in progesterone-treated oocytes shortly before germinal vesicle breakdown (GVBD) or immediately following maturation-promoting factor (MPF) injection. Analysis of phosphoprotein from 32Pi-labeled oocytes by both equilibrium and nonequilibrium two-dimensional gel electrophoresis revealed a large number of qualitative changes in phosphoproteins at GVBD, including both phosphorylation and dephosphorylation events. Time-course studies demonstrated that some of the new phosphoproteins appeared as early as 0.36 GVBD50, and all changes were stable at least through GVBD. The pattern of new phosphoproteins at GVBD was similar in oocytes microinjected with a partially purified preparation of MPF. A number of the new phosphoproteins were heat stable, which may facilitate their purification and characterization. These results support the hypothesis that key regulatory events during oocyte maturation are controlled by protein phosphorylation.     

Changes in protein synthesis pattern during in vitro maturation of goat oocytes.

The regulation of meiotic events of goat oocytes from prophase I to metaphase II was studied by inhibiting protein synthesis at different times of the transition and by analyzing the changes in the protein synthesis pattern during maturation. Protein synthesis was required for germinal vesicle breakdown (GVBD). Nevertheless, the concomitant event to the rupture of germinal vesicle, i.e., chromosome condensation, took place even in a cycloheximide-containing medium. The transition from metaphase I to metaphase II was also protein synthesis dependent as evidenced by experiments using this protein synthesis inhibitor. The inhibition was partly reversible, i.e., after removal of the drug, oocytes were able to progress until metaphase I but could not proceed beyond this stage. Changes in the protein synthesis pattern were studied by radiolabelling of oocytes with [35S]methionine. These changes were correlated with the nuclear status of the oocyte: At GVBD, a polypeptide of 25 kD disappeared, while one of 27 kD appeared. At the same time, a polypeptide of 33 kD appeared, whereas concomitantly one of 34 kD became barely detectable and finally disappeared as the maturation progressed. During maturation, the synthesis of a 67 kD polypeptide increased and became predominant at the end of the maturation process. The synthesis of actin decreased after 18 hr of culture from a very high to a low level of synthesis.     

Meiotically incompetent and competent goat oocytes: timing of nuclear events and protein phosphorylation

The ability of mammalian oocytes to resume meiosis and to complete the first meiotic division is acquired sequentially during their growth phase. The acquisition of meiotic competence in goat oocytes has been previously correlated with follicular size (9). Since protein phosphorylation/dephosphorylation play a key role in oocyte maturation, it could be that in meiotically incompetent oocytes, such post-translational modifications are inadequate. The aim of this study was to analyze whether changes in oocyte proteins phosphorylation occurred during the acquisition of meiotic competence. For this propose, goat oocytes were divided into 4 classes according to follicular size and meiotic competence: Class A oocytes from follicles < 0.5 mm in diameter: Class B oocytes from follicles 0.5-0.8 mm; Class C oocytes from follicles 1-1.8 mm and class D oocytes from follicles > 3 mm. The protein phosphorylation patterns of these classes of oocytes were studied at different times of in vitro maturation. After 4h of culture, when all oocytes were in the germinal vesicle stage, only the oocytes from Class D displayed the phosphoproteins at 110 kD, 31 kD and around 63 kD. In contrast to Class D oocytes Classes B and C oocytes were partially competent to mature, they underwent germinal vesicle breakdown later than fully competent Class D oocytes and remained in early prometaphase I or in metaphase I, respectively. They exhibited the phosphoprotein changes that are associated with commitment to resume meiosis; but the changes occurred later than in Class D oocytes, which were fully competent to reach metaphase II. After 27 h of culture, the phosphorylation patterns of Class B, C and D oocytes were identical, whereas the meiotic stages reached were quite different. The phosphoprotein changes associated with oocyte maturation did not occur in meiotically incompetent Class A oocytes, which were blocked at the germinal vesicle stage. From these results it can be concluded that, at the GV stage, meiotically incompetent and competent goat oocytes display different patterns of protein phosphorylation. Once oocytes are able to resume meiosis they undergo specific phosphorylation changes, but whether these changes are markers or regulators of maturation events remains to be determined.     

Analyses of mitogen-activated protein kinase function in the maturation of porcine oocytes

The function of mitogen-activated protein kinase (MAPK) during porcine oocyte maturation was examined by injecting oocytes with either mRNA or antisense RNA of porcine c-mos protein, an upstream kinase of MAPK. The RNAs were injected into the cytoplasm of porcine immature oocytes immediately after collection from ovaries, then the oocytes were cultured for maturation up to 48 h. The phosphorylation and activation of MAPK were observed at 6 h after injection of the c-mos mRNA injected-oocytes, whereas in control oocytes, MAPK activation was detected at 24 h of culture. The germinal vesicle breakdown (GVBD) rate at 24 h of culture was significantly higher in c-mos mRNA-injected oocytes than in control oocytes. In contrast, although injection of c-mos antisense RNA completely inhibited phosphorylation and activation of MAPK throughout the maturation period, the GVBD rate and its time course were the same in noninjected oocytes. The degree of maturation-promoting factor (MPF) activation was, however, very low in oocytes in the absence of MAPK activation. Most of those oocytes had both abnormal morphology and decondensed chromosomes at 48 h of culture. These results suggest that MAPK activation is not required for GVBD induction in porcine oocytes and that the major roles of MAPK during porcine oocyte maturation are to promote GVBD by increasing MPF activity and to arrest oocytes at the second metaphase.     

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.     

Cyclin B1 turnover and the mechanism causing insensitivity of fully grown mouse oocytes to cycloheximide inhibition of meiotic resumption

Cyclin B1 turnover and the insensitivity of fully-grown mouse oocytes to cycloheximide (CHX) inhibition of germinal vesicle breakdown (GVBD) were examined by assaying GVBD and cyclin B1 levels after treatment of oocytes with various combinations of eCG and CHX. Whereas over 95% of oocytes underwent GVBD after culture for 24 h with CHX alone, only 10% did so after culture with CHX + eCG (P < 0.05). In addition, preculture with eCG alone had no effect, but preculture with eCG + CHX prevented GVBD during a second culture with CHX alone. Therefore, we inferred that eCG delayed GVBD long enough for CHX inhibition of protein synthesis to allow cyclin B1 to decrease below a threshold where GVBD became dependent upon its de novo synthesis. However, western blot revealed no cyclin B1 synthesis, but cyclin B1 degradation, as long as GVs were maintained intact with eCG. Regarding the function of CHX in preculture without protein synthesis to block subsequent GVBD, whereas eCG delayed GVBD for only 3 h, CHX had an ongoing effect that further postponed GVBD, thus allowing cyclin B1 to decrease below the threshold. When oocytes precultured with eCG + CHX were further cultured without eCG and CHX, cyclin B1 first decreased but then, because of the ongoing effects of CHX, increased to a level sufficient to induce GVBD. The content of P34Cdc2 was not altered under any of the culture conditions (P > 0.05). We concluded that insensitivity of mouse germinal vesicle (GV) oocytes to CHX was due to the presence of sufficient cyclin B1, and that cyclin B1 level in such oocytes was maintained by an equilibrium between synthesis and degradation.     

Inhibition of protein synthesis affects histone H1 kinase,but not chromosome condensation activity,during the first meiotic division of pig oocytes

The influence of protein synthesis on the regulation of the first meiotic division was studied in pig oocytes. We show that histone H1 kinase activity gradually increases during in vitro culture of pig oocytes, reaching maximum in metaphase I stage after 24 hr of culture. However, in the presence of the protein synthesis inhibitor cycloheximide, histone H1 kinase is not activated during the whole culture period, and after 24 hr it is approximately at the same level as in prophase-stage oocytes. The gradual increase in phosphorylation of six proteins of molecular weights 39, 48, 53, 66, 96, and 120 kDa, observed during the first 24 hr of culture, was not detected when cycloheximide was added to the culture medium. Similarly, the decrease in phosphorylation of a 90-kDa protein was not seen in cycloheximide-treated oocytes. On the other hand, the levels of both MPF components, p34^cdc2 and cyclin B, which were found to be nearly constant during the first meiotic division, were not influenced by cycloheximide treatment as revealed by Western blotting. The process of germinal vesicle breakdown (GVBD) was totally blocked by cycloheximide. The condensation of chromatin, however, was not influenced, suggesting that GVBD and chromosome condensation could be regulated independently. The different degrees of MPF activation involved in these processes, as well as the nature of the protein(s) which must be synthesized for triggering GVBD, are discussed. © 1995 Wiley-Liss, Inc.