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)     

Prepubertal bovine oocyte: A negative model for studying oocyte developmental competence

To identify potential markers of maturation quality, differences in developmental capacity between cow and calf oocytes were compared in parallel with their constitutive and neosynthetic protein profiles before and after in vitro maturation (IVM). A comparison was also made between the protein profiles of follicular fluid (FF) from calf and cow ovaries. The effect of epidermal growth factor (EGF) during IVM on the subsequent development of prepubertal calf oocytes was examined. The effect of the presence of fetal calf serum (FCS) during development of embryos originating from calf oocytes was also examined. No differences were noted between the constitutive proteins of cow and calf oocytes and only a minor modification was observed before IVM in the pattern of neosynthesized proteins (presence of a band of 37 kD and a slight increase in the intensity of band of 78 kD in cow as compared to calf oocytes). However, the comparison of constitutive protein profiles from calf and cow FF demonstrated quantitative (the bands of 34 and 45 kD were more intense for cow than for calf) differences. EGF receptors (EGF-R) were demonstrated on cumulus—oocytes complexes (COCs) by immunofluorescence. There was no difference in intensity between cow and calf COCs. Furthermore, the addition of EGF during IVM of calf oocytes dramatically stimulated cumulus expansion and significantly increased the cleavage rate at 72 h post-insemination (82% vs 67%), as well as the proportion of embryos at the 5- to 8-cell stage at this time (54% vs 43%). Also, blastocyst yields at day 6 (11% vs 5%) and at day 8 (17% vs 10%) were significantly higher in the presence of EGF P < 0.05). The addition of FCS to synthetic oviduct fluid droplets at day 2 of culture (48 hpi) had no effect on cleavage, blastocyst yield, or blastocyst cell number. In conclusion, differences in developmental ability between calf and cow oocytes would appear to be not solely linked to differences in oocyte protein patterns. It is likely that the FF, which constitutes the microenvironment in which the oocyte develops, plays a major modulating role in determining the fate of the oocyte/follicle. © 1996 Wiley-Liss, Inc.     

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.     

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.     

Effects of cyclic AMP on the spontaneous meiotic maturation of cumulus-free bovine oocytes cultured in chemically defined medium

The rate of spontaneous meiotic maturation and the period of commitment to this process were determined in bovine oocytes devoid of surrounding cumulus cells, cultured in chemically defined medium with bovine serum albumin in the absence of serum. The effects of compounds that are known to elevate levels of intracellular cyclic adenosine monophosphate (cAMP) on the resumption and progression of meiosis were investigated. Bovine oocytes were mass-harvested, denuded of cumulus cells, and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin. Intracellular cAMP levels were indirectly modified using 8-bromo-cAMP, dibutyryl cAMP (dbcAMP), forskolin, or 3-isobutyl-1-methyl xanthine (IBMX). Meiotic maturation was scored cytogenetically. Ninety percent of denuded bovine oocytes mature after 24 h, with 65% progressing beyond anaphase I. These oocytes remain at the germinal vesicle (GV) stage for up to 8 h in culture. GV breakdown (GVBD) occurs in 40.5% of oocytes at 9 h. The peak times for the different meiotic stages were 12 h for diakinesis, 15 h for late diakinesis to metaphase I, 20 h for metaphase I, and 24 h for telophase I. By 48 h, most had reached metaphase II. There is a 2-h lag period between the time at which they become irreversibly committed to mature (at 7 h) and when they demonstrate GVBD (at 9 h). Incubation for 12 h with high concentrations of 8-bromo-cAMP and forskolin significantly inhibited GVBD, while the effect of dbcAMP was similar but less pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect of ovarian cortex cells on nuclear maturation of sheep oocytes during in vitro maturation

The objective of this study was to investigate the influence of ovarian cortex cells (OCCs) monolayers on the nuclear maturation of sheep oocytes with or without cumulus cells during IVM. Sheep ovaries collected from a local abattoir were transported to the laboratory in warm PBS containing antibiotics within 2-3h after collection. Cumulus-oocyte complexes (COCs) were obtained by aspiration and evaluated in a pre-incubated Hepes-modified TCM 199 medium. The selected COCs were randomly divided into six treatment groups: group 1 (control group): oocytes enclosed by cumulus cells were cultured in maturation medium; group 2 (co-culture group): oocytes enclosed by cumulus cells co-cultured with OCCs monolayers; group 3 (conditioned group): oocytes enclosed by cumulus cells were cultured in OCCs-conditioned medium; group 4 (denuded group): denuded oocytes were cultured in the maturation medium; group 5 (denuded co-culture group): denuded oocytes co-cultured with OCCs monolayers in maturation medium; group 6 (denuded conditioned group): denuded oocytes were cultured in OCCs-conditioned medium. After maturation for 24h, the oocytes in each treatment group were fixed, stained and the nuclear status of the oocytes were assessed under an inverted microscope. The highest percentage of metaphase II (M-II) stage oocyte was observed in group 2 (86.3%) and the lower percentage was observed in the denuded groups (group 4-6). The removal of cumulus cells dramatically decreased the percentage of M-II stage oocyte. The comparison of the nuclear maturation status in group 4-6 showed that the co-culture of oocyte with OCCs monolayers resulted in progression to completing the GVBD stage to reach the M-II stage. The results demonstrated that the presence of OCCs could positively influence the meiotic resumption and progression of sheep oocytes during IVM.     

Biochemical and developmental evidence that ooplasmic maturation of prepubertal bovine oocytes is compromised

Our previous studies have shown that oocytes collected from prepubertal calves lack developmental competence. The overall objective of this study was to assess causes by comparing biochemical and physiologic changes during in vitro maturation of oocytes collected from ovaries of adult cattle at slaughter and from superstimulated calves (<6 mo old) by either laporotomy or ultrasound-guided follicular aspiration. Activity and/or concentrations of maturation-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and inositol 1,4,5-trisphosphate receptor (IP(3)R) were determined by measuring phosphorylation of histone H-1 kinase, phosphorylation of myelin basic protein, or Western blotting, respectively, and were compared between oocytes collected from calves and for those collected from cows. The activities of MPF and MAPK and the relative amount of IP(3)R were significantly lower in calf oocytes. The physiologic significance of these observations was determined by assessing the developmental potential of embryos derived by reciprocal transfer of metaphase II (M-II) chromosomes between cow and calf ooplasts and transfer of adult cumulus cells (G0/G1) into cow and calf ooplasts. Procedural controls consisted of transfer of M-II between adult oocytes and parthenogenic activation of adult and calf oocytes. Adult parthenogenically activated oocytes cleaved and developed to blastocysts at a higher rate than did similarly activated calf oocytes (42.1% vs. 3.4%, P < 0.05). Cleavage was also higher in reciprocal M-II transfer embryos containing adult ooplasm (46.2% vs. 12.0%, P < 0.05). Cleavage (66.7% vs. 21.9%, P < 0.05) and development to blastocyst (20.1% vs. 4.8%, P < 0.05) of nuclear transfer embryos reconstructed from adult cumulus cells was higher after transfer to adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation.     

Effect of transport and storage temperature of ovaries on in vitro maturation of bitch oocytes

In this study, the effects of ovary transport and storage temperature on in vitro maturation of bitch oocytes were investigated. Ovaries were collected from 23 mature bitches and one randomly selected ovary of each pair (n=23 pairs) was transported in physiologic saline at 4 degrees C, while the other one at 35-38 degrees C for 2-4h. A total of 316 cumulus oocyte complexes (COCs) were obtained from the 4 degrees C group and 301 COCs from the 35-38 degrees C group. All COCs were matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), essential and non-essential amino acids at 38 degrees C in a humidified 5% CO2, 5% O2, and 90% N2 atmosphere for 72 h. At the end of the in vitro maturation period, nuclear maturation of oocytes was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), undetermined nuclear maturation (UDNM), and MI+MII. The nuclear maturation rates to MI, MII, and MI+MII stages were 60.44%, 10.75%, and 71.20% in the 4 degrees C group and 37.20%, 7.64%, and 45.85% in the 35-38 degrees C group, respectively. The data demonstrated that oocytes obtained from ovaries transported at 4 degrees C had higher maturation rates than from the ones transported at 35-38 degrees C (p<0.001).     

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 2. The timing of in vitro nuclear oocyte maturation

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h intervals (0-27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7+/-0.4 and 24.8+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine 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.     

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.     

Leucine uptake and incorporation at three stages of mouse oocyte maturation

Mouse oocytes at the dictyate, metaphase I and metaphase II stages of maturation were incubated with and without cumulus cells in ¹⁴C-leucine (1.7 mM, spec. act. = 35 μCi/μmole) for a 1 h pulse after which the total amount of radioactivity taken up during this time and the amount present in the acid-insoluble fraction were measured. In the absence of cumulus cells there was a peak in total leucine uptake at metaphase I. Cumulus cells resulted in increased oocyte uptake at the dictyate stage, decreased uptake at metaphase I with no effect at metaphase II. The variations in uptake with maturation stage and cumulus cells were not paralleled by similar changes in the acid-insoluble radioactivity. There was no change in the acid-insoluble incorporation with progressive maturation, but there was a positive effect of cumulus cells at the dictyate and metaphase I stages. Small and large dictyate oocytes took up and incorporated similar amounts of leucine even though the smaller oocytes did not mature in vitro. The results are discussed in relation to the changing association between the oocyte and cumulus cells with progressive maturation.     

Time-dependent effects of cycloheximide and alpha-amanitin on meiotic resumption and progression in bovine follicular oocytes

To identify the stage during maturation at which new protein and RNA are synthesized for meiotic resumption, follicular oocytes were cultured in TCM-199 with the protein synthesis inhibitor cycloheximide or the hnRNA synthesis inhibitor alpha-amanitin. Although the meiotic resumption of cumulus-enclosed oocytes was completely blocked by the addition of 25 microg/ml cycloheximide at 4 h after the onset of culture, 23% of oocytes cultured from 5 h post cultivation in the medium with cycloheximide underwent germinal vesicle breakdown (GVBD). By further delaying the addition of cycloheximide, the proportion of oocytes which underwent GVBD increased. Addition of the inhibitor at 8 h or more post cultivation resulted in GVBD occurring in more than 87% of oocytes, though none of them were able to proceed beyond the metaphase I stage. In contrast, the addition of 50 microg/ml alpha-amanitin from the onset of culture significantly reduced the proportion of GVBD to 75% in cumulus-enclosed oocytes, while no significant reduction in the proportions of GVBD was noted in the case of its addition from 1 h of culture onward. However, denuded oocytes were almost insensitive to any treatments with alpha-amanitin. These results indicate that protein synthesis in the oocytes and RNA synthesis in the cumulus cells soon after the onset of culture are necessary for GVBD and that continuous protein synthesis following GVBD is indispensable for progression of the meiotic division in bovine oocytes.     

Temperature-induced abnormalities in sheep oocytes during maturation

The susceptibility of sheep oocytes to temperature changes during maturation in vitro was tested by reducing the incubation temperature to 20 degrees C at various stages of meiosis. Cooling induced chromosomal abnormalities including disorganized metaphase plates and multipolar spindles in 28-54% of oocytes cooled at all stages of meiosis from germinal vesicle breakdown (GVBD) to metaphase II. The time of GVBD (8-11 h after the start of culture) was the most sensitive to cooling, whereas fewest abnormalities were found in oocytes cooled in late metaphase I (16-19 h). In addition to the chromosomal abnormalities, unusual vesicles appeared in the cytoplasm of oocytes cooled at 8-11 h and 12-15 h. No abnormalities in protein synthesis were detected by one-dimensional SDS gel electrophoresis. The consequences of the abnormalities for the developmental potential of the cooled oocytes were tested by transfer to recipient ewes and fertilization in vivo. After 12 days of development only 6% and 11% oocytes cooled at 12-15 h and 20-23 h respectively had developed to expanded blastocysts, compared with 44% of control oocytes. The results demonstrated that maturing sheep oocytes are very sensitive to a drop in temperature.     

The fall of biological maturation promoting factor (MPF) and histone H1 kinase activity during anaphase and telophase in mouse oocytes.

Cell fusions have been used to determine the biological activity of the MPF complex in murine oocytes during their progression through anaphase and telophase to metaphase II. Oocytes (1) at metaphase I, (2) during the anaphase-telophase transition, or (3) at metaphase II were fused to germinal vesicle-staged (immature) oocytes. The hybrids were cultured for 1 h in the presence of db cAMP before fixation and nuclear evaluation. Metaphase I oocytes invariably induced germinal vesicle breakdown (GVBD) in the immature partner. By contrast, anaphase/telophase oocytes never induced GVBD in immature oocytes. The capacity to induce GVBD reappears after the formation of the second metaphase plate. In a second study, histone H1 kinase activity was measured during mouse oocyte maturation in single oocytes. H1 kinase activity was low in GV oocytes, increased sharply at MI, declined during anaphase and telophase and increased again at MII. After egg activation, H1 kinase activity was reduced to basal levels. These results provide direct evidence that a drop in activity of MPF in murine oocytes occurs concomitantly with the exit from metaphase I; MPF activity remains low until the cell re-enters metaphase.     

Selection of follicles, preculture oocyte evaluation, and duration of culture for in vitro maturation of equine oocytes

Equine oocytes (n = 537) were collected from slaughterhouse ovaries (n = 118 mares) by scraping the internal follicular wall. Preculture record was made of the appearance of oocyte investments (no cumulus, corona radiata only, compact cumulus, expanded cumulus), appearance of cytoplasm (homogeneous, condensed heterogeneous/fragmented), and nuclear maturation stages (germinal vesicle, germinal-vesicle breakdown, metaphase I, metaphase II, degenerated). There was no difference between follicles > 30 mm and follicles < or = 30 mm in the preculture frequency distribution among the 5 nuclear stages; 96% were at either the germinal vesicle or germinal-vesicle breakdown stages. Oocytes from follicles 5 to 30 mm were cultured in modified TCM-199 for 18, 24, 36 and 48 h. Postculture nuclear maturation classifications were immature (germinal vesicle, germinal-vesicle breakdown, and metaphase I), mature (metaphase II or secondary oocyte), and degenerated. The frequency distribution of oocytes among the 3 postculture maturation classifications changed (P < 0.05) at 18 h (15% mature oocytes), changed (P < 0.05) further at 24 h (55% mature oocytes), with no additional change for 36 or 48 h. The only preculture cytoplasm group that affected the postculture results was the heterogeneous/fragmentation group which had a high proportion of postculture degenerated oocytes (67%); however, only 4% of oocytes were in this group. Luteal status of the mare had an effect (P < 0.05) on the frequencies of the maturation classifications, but not enough to be useful in selecting oocytes. Consistency of the follicle and the type of oocyte investment did not alter significantly the maturation frequencies. The frequency of degenerated oocytes after culture was high under the following conditions: 1) diameter of the follicle from which the oocyte was selected was 5 to 10 mm (44% degenerated oocytes), 2) the largest follicle per pair of ovaries was < or = 10 mm (63%), and 3) the mare was pregnant (66%). These results were probably related to the reported high frequency of atretic follicles in the 5- to 10-mm population. In summary, oocytes from individual follicles < or = 10 mm or from follicles in which the largest follicle per mare was < or = 10 mm were the poorest candidates for in vitro maturation.     

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.