Resumption of meiosis in pig oocytes cultured with cumulus and parietal granulosa cells: the effect of protein synthesis inhibition

In denuded and cumulus-enclosed pig oocytes, puromycin at concentrations 5, 10, and 25 micrograms/ml did not lower the rate of germinal vesicle breakdown (GVBD) after 24 h of culture. GVBD was prevented in 50, 75, and 100 micrograms/ml of puromycin. After 40 h of culture, 5 and 10 micrograms puromycin/ml impaired significantly incidence of metaphase II (42 and 30%), respectively. Concentrations of 25 and 50 micrograms puromycin/ml absolutely prevented the first polar body (I PB) expulsion. The results indicated that GVBD in pig oocytes is far less sensitive to puromycin than I PB expulsion. Culture of cumulus-enclosed pig oocytes isolated with a piece of membrana granulosa (C + P oocytes) did not allow GVBD after 24 and 32 h in control medium. After 24 h of culture, GVBD occurred in 43 and 56% of C + P oocytes in the medium supplemented with 17 and 25 micrograms puromycin/ml. GV was broken down in 80 and 68% of C + P oocytes cultured in 17 and 25 micrograms puromycin/ml for 32 h. It is concluded that inhibition of protein synthesis by puromycin released pig oocytes from the block exerted by granulosa cells.     

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.     

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.     

Cytochalasin D treatment induces meiotic resumption in follicular sheep oocytes

Ovine cumulus-enclosed oocytes collected from antral follicles (3-5 mm in diameter) were cultured in vitro with 2 x 10(6) granulosa cells/ml in the presence or absence of gonadotropins or in the presence of cytochalasin D (CD). The maturation rate was assessed after 24 h of culture. In the control group, in the presence of gonadotropins (follicle-stimulating hormone-luteinizing hormone (FSH-LH; -10 micrograms/ml) 100% of the oocytes reached metaphase II. Whereas intercellular junctions were no longer present after 6-7 h of culture, germinal vesicle breakdown (GVBD) occurred by the same time. In contrast, in the absence of gonadotropin, the majority of the oocytes (59%) remained blocked in GV stage. The inhibition exerted by the granulosa cells on meiotic resumption was overcome when the cumulus-oocyte complexes (COCs) were incubated in CD (5 micrograms/ml) for 6 h at the beginning of the culture. Under these conditions, 85% of the oocytes matured with extrusion of the first polar body. Cytological analysis by cytofluorescence (NBD phallacidin) and electron microscopy showed that, after 6 h of treatment, CD provoked a redistribution of the microfilaments, mainly in the cumulus cells and to a lesser extent in the oocyte cortex. Intercellular junctions disappeared concomitantly with a significant decrease of the intercellular transport of tritiated uridine. The initiation of GVBD occurred at the same time. These results indicate that the resumption of meiosis was correlated with a loss of both junctional complexes (intermediate and gap junctions) between the cumulus cells and the oocyte.     

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.     

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.     

Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes

In cows, protein synthesis is required for germinal vesicle breakdown (GVBD). This study examines more closely the need for protein synthesis and the nuclear changes in the bovine oocyte during 24 h of culture. Bovine oocytes with compact and complete cumulus were washed and incubated in groups of 10 for up to 24 h in 50-microliters drops of TCM-199 supplemented with follicle-stimulating hormone (NIAMADD, 0.5 micrograms/ml), luteinizing hormone (LH) NIAMADD, 5 micrograms/ml), estradiol-17 beta (1 microgram/ml), pyruvate (20 microM), and 10% heat-treated fetal calf serum. Medium was overlaid with paraffin oil. Oocytes (n = 891) were fixed at the end of each 3-h interval from 0 to 24 h of culture, or at 24 h after addition of cycloheximide (10 micrograms/ml at 10 different times during maturation (0, 1, 2, 3, 6, 9, 12, 15, 18, 21 h; n = 175). At each time point, the chromosomal status of oocytes was evaluated, frequencies were computed, and the time spent on each step was determined. The germinal vesicle (GV) was present from 0 to 6.6 h, GVBD at 6.6 to 8.0 h, chromatin condensation at 8.0 to 10.3 h, metaphase I at 10.3 to 15.4 h, anaphase I at 15.4 to 16.6, telophase I at 16.6 to 18.0 h, and metaphase II at 18.0 to 24 h. Cycloheximide blocked oocyte maturation at GVBD, if added from 0 to 3 h; at chromatin condensation, if present from 6 to 24 h; and at metaphase I, when present from 9 to 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein modification by phosphorylation during the process of nuclear membrane dissolution in puromycin-treated mouse oocytes.

The present study was undertaken to elucidate the mechanism of nuclear membrane dissolution (NMD) in puromycin-treated mouse oocytes. Treatment of germinal vesicle breakdown (GVBD) oocytes with puromycin (50 micrograms/ml) induced chromosome decondensation with formation of a polar body; these are designated nuclear membrane (NM) oocytes. After withdrawal of puromycin, NM oocytes underwent NMD (approximately 70%) during a 12-h culture period. Either dibutyryl cyclic AMP (dbcAMP, 25-100 micrograms/ml) or isobutylmethylxanthine (IBMX, 0.1-1.0 mM) inhibited the process of NMD in a dose-dependent manner, suggesting the involvement of cAMP in the process of NMD. To determine which protein(s) participated in the transition from interphase to metaphase II during NMD, NM oocytes were labeled with [35S]methionine, and one- and two-dimensional gel electrophoresis were performed. Although the synthesis of stage-specific proteins during NMD was not found, two specific proteins of Mr 27,000 and 46,000, which were synthesized at interphase following removal of puromycin, were modified during NMD. Phosphatase treatment and 32PO4-labeling experiments indicated that phosphorylation was responsible for these modifications, which were inhibited by either dbcAMP or IBMX. Therefore, it appears that phosphorylation of specific proteins may play an important role in the transition from interphase to metaphase II.     

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.     

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.     

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.     

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.     

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.     

Chromosome condensation in pig oocytes: lack of a requirement for either cdc2 kinase or MAP kinase activity

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases (cdk), is shown to inhibit germinal vesicle breakdown (GVBD) in pig oocytes. Oocytes treated with 100 microM BL I were arrested in the germinal vesicle (GV)-stage and displayed low activity of cdc2 kinase and MAP kinase. Nevertheless, chromosome condensation occurred and highly condensed bivalents were seen within an intact GV after a 24-hr culture in the presence of BL I. The inhibitory effect of BL I on MAP kinase activation during culture was likely mediated through a cdk-dependent pathway, since MAP kinase activity present in extracts derived from metaphase II eggs was not inhibited by BL I. The block of GVBD could be released by treating oocytes with okadaic acid (OA), an inhibitor of type 1 and 2A phosphatases; 82% of the oocytes treated with the combination of OA/BL I underwent GVBD, and MAP kinase became activated, while cdc2 kinase remained inhibited. These results suggest that both chromosome condensation and GVBD could occur without activation of cdc2 kinase, whereas an increase in MAP kinase activity may be a requisite for GVBD in pig oocytes in conditions when cdc2 kinase activation is blocked by BL I.     

Heparin effect on in vitro nuclear maturation of bovine oocytes

Oocytes undergo numerous biochemical and morphological changes during their development from preantral to preovulatory phases. In vitro studies have suggested several compounds that might induce oocyte maturation. Heparin is a natural component of ooplasm, follicular fluid and uterine fluid and previous studies indicated that it might act as a chromatin maturation factor in bovine oocytes. We tested this hypothesis in vitro by timing germinal vesicle breakdown (GVBD) and first polar body (PB) formation without any other natural or introduced factors that might influence the rate of oocyte maturation. We also determined if these oocytes could be fertilized. Bovine oocytes were incubated in a salt medium and TCM 199 supplemented with different concentrations of heparin for 24 h at 37.5 degrees C in a humidified atmosphere of 5% CO2. With 1.0 and 6.5 mg/ml heparin, the time of GVBD was reduced from 4.7+/-1.1 h to about 1.5 h and the time of first PB formation was reduced from 22.0+/-1.1 h to 9.0-11.0 h in salt medium. In TCM 199, only 6.5 mg/ml heparin significantly reduced the time of PB formation. In both incubation media, 1.0 and 6.5 mg/ml heparin induced GVBD, extrusion of the first PB and formation of the metaphase II nucleus. Moreover, heparin did not interfere with the fertilization of oocytes matured in TCM 199. Based on the results, we propose that heparin plays an important role in the rearrangement of the oocyte chromatin and acts as an oocyte maturation factor.     

Nuclear staining and culture requirements for in vitro maturation of domestic bitch oocytes

Oocyte nuclear staining and culture requirements for in vitro maturation (IVM) in the bitch have yet to be fully investigated. In the first part of this study we investigated 7 methods for labeling nuclear material (573 oocytes). The most favorable method involved fixation plus aceto-orcein staining and light microscopy. The influence of serum supplementation of the culture medium for IVM was then investigated (1292 oocytes). Culture was performed in media supplemented with no serum or with 5, 10 and 20% fetal calf serum (FCS) and 0.3 or 4% bovine serum albumin (BSA). Identifiable nuclear material was either a germinal vesicle (GV) or GV breakdown (GVBD). After 48 h in medium plus 0, 5, 10 or 20% FCS and 0.3 or 4% BSA, the percentage of oocytes matured to GVBD was 13, 9, 15, 23, 36 and 40%, and the percentage matured to metaphase I/anaphase I/metaphase II was 4, 12, 24, 14, 36 and 13%, respectively. After 96 h, maturation to GVBD was 31, 14, 21, 11, 50 and 38%, and to metaphase I/anaphase I/metaphase II it was 6, 5, 3, 19, 15 and 9%, respectively. Within the limits of this study, BSA or high concentrations of FCS appear to be optimal for bitch oocyte maturation in vitro.     

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.     

The role of RhoA in the germinal vesicle breakdown of mouse oocytes

We have investigated a new role of RhoA in the germinal vesicle breakdown (GVBD) of mouse oocytes. First, RhoA was identified by immunostaining and ADP-ribosylation in germinal vesicle (GV) stage-oocytes. RhoA was mainly localized in the ooplasmic area, but rarely detected in germinal vesicle. Incubation of oocyte extract with C3 transferase induced a strong ADP-ribosylation at about 25 kDa. Incubation of GV-stage oocytes in culture medium induced the spontaneous maturation to GVBD by about 78 and 87% of total oocytes at 1 and 3 h, respectively. However, microinjection of C3 transferase into GV-stage oocytes significantly inhibited GVBD at 1 (GVBD = 29%) and 3 h (GVBD = 49%). To study the role of reactive oxygen species (ROS) in the oocyte maturation, the level of intra-oocyte ROS was measured using a ROS-specific fluorescent dye H(2)DCFDA during the oocyte maturation. Spontaneous maturation of GV-stage oocytes induced a significant increase of ROS at 3 h by about twofold over the control level and then the increased level was maintained until 6 h. However, microinjection of C3 transferase inhibited the production of intra-oocyte ROS. Incubation with ROS scavengers, N-acetyl-l-cysteine and catalase, blocked the ROS increase. The ROS scavengers also significantly inhibited GVBD, as did C3 transferase. Thus, it was proposed that RhoA was involved in the GVBD, possibly by the production of ROS in mouse oocytes.     

Kinetics of meiotic progression, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes: species specific differences in the length of the meiotic stages

The kinetics of nuclear maturation, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes were examined. A further objective was to determine the duration of the meiotic stages during the maturation process. Porcine and bovine cumulus-oocyte complexes (COCs) were incubated in TCM 199 supplemented with 20% (v/v) heat inactivated fetal calf serum (FCS), 0.05microg/ml gentamycin, 0.02mg/ml insulin, 2.5microg/ml FSH and 5microg/ml LH. COCs were removed from the culture media in hourly intervals starting immediately after recovery from the follicle until 24 (bovine) or 48h (porcine) of culture. Oocytes were either fixed to evaluate the maturation status or the activity of MPF, assessed by its histone H1 kinase activity, and MAP kinase were determined by a radioactive assay simultaneously. In oocytes of both species, the MPF activity oscillated during the culture period with two maxima corresponding with the two metaphases: between 27-32 and after 46h (porcine) and between 6-9 and after 22h (bovine). There was a temporary decline in activity after 33-38 (porcine) and after 19h (bovine), which corresponded with anaphase I and telophase I. MAP kinase activity increased during the whole culture period and reached maximum levels after 47 (porcine) and after 22h (bovine). In porcine oocytes, the MAP kinase was activated before GVBD and MPF activation. In bovine oocytes, MPF and MAP kinase were activated at approximately the same time as the GVBD (8-9h of incubation). In average porcine, oocytes remain 23.4h in the germinal vesicle (GV) stage (13h in GV I, 5.7h in GV II, 3.2h in GV III and 1.5h in GV IV), 0.9h in diakinese, 9.6h in the metaphase I, 2.8h in anaphase I and 1.9h in telophase I of the first meiotic division. In bovine oocytes, the temporal distribution of the meiotic stages were 8.5h for the GV stage, 1.2h for diakinese, 8.3h for metaphase I, 1.6h for anaphase I and 1.9h for telophase I. These results indicate that the duration of the meiotic stages differs between the species and that MAP kinase is activated before MPF and GVBD in porcine oocytes.