Meiotic inhibition with different cyclin-dependent kinase inhibitors in bovine oocytes and its effects on maturation and embryo development

Cyclin-dependent kinase inhibitors (CDKIs) such as butyrolactone I (BL-I) and roscovitine (ROS) maintain bovine oocytes blocked at the germinal vesicle (GV) stage. Bohemine (BOH), another CDKI, has been used for oocyte activation. The objective of this study was to determine whether BOH blocks meiosis and to compare its efficiency with other CDKIs (ROS and BL-I). Oocytes were cultured for 24 h in 0, 50, 100 and 150 microM BOH to determine the best concentration for blocking meiosis (experiment 1). GV rates were 3.3%, 64.5%, 83.3% and 88.9% (0,50, 100 and 150 microM, respectively). Experiment 2 compared meiotic inhibition efficiency of BOH (100 microM), ROS (25 microM) and BL-I (100 microM). BL-I presented the highest GV rates (97.5%). BOH and ROS were similar to each other (85.4% and 79.9%, respectively). To assess the reversibility of meiotic inhibition (experiment 3), oocytes underwent in vitro maturation (IVM) for 18 h after the 24 h inhibition. Control oocytes were submitted to IVM for 18 h (C18) or 24 h (C24). Maturation rates were either similar to (ROS and BL-I: 96.0% and 93.6%, respectively) or superior to (BOH, 96.9%) C24 (91.0%). All groups were superior to C18 (82.5%). In experiment 4, oocytes were treated as in experiment 3 and then in vitro fertilized and cultured for 8 days. Blastocyst rates for BL-I (32.3%) were similar to C24 (35.0%), while those for BOH (20.2%) and ROS (24.2%) were inferior. All groups were inferior to C18 (43.4%). The results show that: (a) BOH inhibits meiosis resumption; (b) BL-I is the most effective of the CDKIs tested for blocking meiosis; (c) culture of oocytes with meiosis inhibitors is fully reversible in terms of nuclear maturation but they may either decrease (BOH and ROS) or maintain (BL-I) embryo development rates.     

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)     

Roles of protein kinase A and C in spontaneous maturation and in forskolin or 3-isobutyl-1-methylxanthine maintained meiotic arrest of bovine oocytes

Four hypotheses were tested using isolated bovine oocytes. (1) Cumulus oocyte complexes (COCs) or denuded oocytes (DOs) were cultured with the protein kinase A (PKA) inhibitor, H-89, to test if meiotic arrest induced by forskolin or IBMX was due to cAMP-stimulated PKA activity or nonspecific effects of these cAMP elevators. (2) COCs were cultured with a protein kinase C (PKC) stimulator (PDDβ) or inhibitor (GF109203x) to test if PKC modulation altered oocyte maturation. (3) COCs were prestimulated for 15 min with (a) PDDβ followed by cotreatment with forskolin, or (b) with H-89 or H-7 followed by cotreatment with GF109203x, to test for interaction between the PKA and PKC signal transduction pathways. (4) H-89 was added to spontaneously maturing COCs at intervals 0–18 hr to test if H-89 interfered with the transition between meiosis I and II. The results were as follows: H-89 interfered with forskolin or IBMX arrested oocytes in a dose-response manner (IBMX ED₅₀ = 41 μM for COCs; forskolin ED₅₀ = 9 μM for denuded oocytes). Prestimulation with PKC induced meiotic resumption in COCs in spite of the presence of forskolin [PDDβ followed by PDDβ + forskolin: 41–47% germinal vesicle (GV) oocytes; forskolin alone: 90–95% GV], while PKC inhibition induced meiotic arrest to a similar extent as forskolin (GF109230x, 85% GV; forskolin, 67–80% GV). Additionally, pretreatment of COCs with H-89 interfered with GF109203x induced arrest (41% vs. 90% GV, respectively). Finally, H-89 interfered with the timely progression of COCs from meiosis I and II. These results indicate that the PKA and PKC pathways can modulate the maturation of bovine oocytes in vitro. © 1996 Wiley-Liss, Inc.     

Alterations and reversibility in the chromatin,cytoskeleton and development of pig oocytes treated with roscovitine

Germinal vesicle (GV) breakdown in mammalian oocytes is regulated by the activation of maturation promoting factor (MPF). We investigated a specific cdc2 kinase inhibitor, roscovitine, to maintain pig oocytes in the GV stage. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 44 hr in NCSU#23 medium containing different levels of roscovitine (0, 10, 20, 30, 40, 50 microM in Experiment 1 and 0, 40, 60, 80, 100, 120 microM in Experiment 2). The COCs were cultured for another 44 hr after removal of the chemical. Twenty oocytes in each group were fixed at 44 hr for immunocytochemical labeling of the cytoskeleton and the rest (approximately 20/group) were fixed at the end of 88 hr after culture. Results showed that the inhibition of the oocyte in the GV stage was not effective when 10-50 microM (Experiment 1) of roscovitine were used (19-34%). When oocytes were released from the inhibitor, similar proportions (70-83%) of oocytes were observed in the MII or advanced stages among treatments. However, when higher concentrations of roscovitine were used (Experiment 2), significantly greater inhibitory effect was observed at the levels of 80-120 microM with 83-91% oocytes being blocked in the GV stage when compared to the control (9%) and the 40-60 microM (27-43%) groups (P < 0.05). Although 15-21% of the oocytes showed abnormal MII morphology with aberrant meiotic spindles and/or formation of cytoplasmic microtubules, a substantial number of oocytes resumed meiosis and reached MII stage at 44 hr after removal of this chemical. In Experiment 3, different concentrations of roscovitine (0, 20, 40, and 80 microM) were tested to examine the length of intervals (0, 11, 22, 33, and 44 hr) for an effective inhibition. Results showed that the inhibitory effect was significantly more prominent at 22 hr than that at 33 and 44 hr after roscovitine treatment in all treatment groups (P < 0.05). This study demonstrated that roscovitine-treated oocytes resumed meiosis after removal of the inhibitor. This could provide flexibility for studying porcine oocyte development and embryo cloning and may have application in other species.     

Kinetics of oocyte maturation and subsequent development of IVF,parthenogenetic, and NT bovine embryos after meiotic inhibition with roscovitine

The developmental competence of bovine oocytes meiotically arrested with specific cdk2 inhibitor roscovitine was studied. After removal of the 32-h block with roscovitine, 82.7 +/- 5.4% reached the metaphase II stage at the end of maturation, which was lower than in controls (96.3 +/- 1.3%, p < 0.001). The process of polar body formation started at 11 h of maturation in the roscovitine group, that is 4 h earlier than in controls and its kinetics was quite similar to controls up to 16 h of maturation, when nearly 70% of oocytes extruded their polar bodies. The rate of blastocyst formation of roscovitine oocytes and their cell number after IVF, parthenogenetic activation, and nuclear transfer (NT) were equal to controls, which demonstrates the possibility of artificially maintaining bovine oocytes in the GV stage for 32 h without altering their preimplantation developmental competence. This approach can be very useful for the management of an NT program where enucleated oocytes are required at specific times or locations.     

绵羊卵母细胞体外核成熟抑制及其对胚胎发育潜力的影响

本研究旨在探讨次黄嘌呤 +dbcAMP或IBMX +dbcAMP对绵羊卵母细胞体外成熟的可逆性抑制作用 ,以及这种抑制作用对卵母细胞胚胎发育潜力的影响。自绵羊卵巢分离卵丘 -卵母细胞复合物进行体外培养 ,培养基中分别加入或不加入上述抑制剂。培养 6h后各组取部分卵母细胞固定染色检查卵母细胞核成熟情况 ;将其余的卵母细胞分别移入无抑制剂的成熟培养基中继续培养 18h后 ,再次检查各组卵母细胞核成熟情况 ,并进行体外受精和胚胎培养。结果表明 :次黄嘌呤 +dbcAMP或IBMX +dbcAM都分别能使 6 0 %以上的绵羊卵母细胞抑制在GV期。这种抑制是可逆性的 ,去除抑制剂后卵母细胞能恢复减数分裂 ,并加快由GVBD到MⅡ的成熟过程。各处理组受精率、卵裂率和囊胚发育率与对照组相比无显著性差异 ,表明卵母细胞的胚胎发育潜力没有受损。上述物质对卵母细胞成熟的可逆性抑制可用于研究卵母细胞成熟及其胚胎发育潜力的调节机制。     

Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation

Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.     

Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes

We investigated the formation of LH receptor (LHR) in cumulus cells surrounding porcine oocytes and the role of LHR in meiotic maturation of oocytes. At least three splice variants of LHR mRNA were detected in cumulus cells, in addition to the full-length form. Low levels of three types of products were seen in cumulus cells from cumulus oocytes complexes (COCs), whereas the full-length form was significantly increased by 12-h cultivation with FSH. The addition of FSH also significantly increased the binding level of biotinylated hCG to COCs. The formation of LHR in FSH-stimulated cumulus cells was not affected by additional 0.5 mM phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the oocytes were synchronized to the germinal vesicle (GV) II stage by exposure to 0.5 mM IBMX and FSH for 20 h. The binding of LH to its receptor induced a further increase in cAMP level and progesterone production and acceleration of meiotic progression to the metaphase I stage. The oocytes cultured with LH for 24 h following cultivation with FSH and IBMX were used for in vitro fertilization. At 6 days after in vitro fertilization, blastocyst rate in oocytes matured under these conditions was significantly higher than that of oocytes cultured in the absence of LH. Treatment of oocytes with FSH and 0.5 mM IBMX to express LH receptor in cumulus cells while holding oocytes at the GV II stage is a very beneficial way to produce in vitro-matured oocytes, which have high developmental competence.     

The influence of cumulus-oocyte complex morphology and meiotic inhibitors on the kinetics of nuclear maturation in cattle

This study evaluated whether pre-established morphological classes of bovine cumulus oocyte complex (COCs) differ in their kinetics of meiosis resumption after 4 h of incubation and whether the timing of COCs resumption of meiosis differed after a period of maintained meiotic arrest. Bovine COCs were aspirated from 2- to 5- mm follicles and classified according to the state of their cumulus cells and cytoplasm (Classes 1 to 3). Groups of 15 to 20 COCs were fixed at 0 h or after an incubation period of 4 h. In addition, COCs from Class 1 were first incubated for 4 h on a theca cell monolayer or in the presence of 2 microg/mL of cycloheximide, rinsed and then incubated in cycloheximide and theca cell-free medium for another 4 h. Oocytes then were fixed and evaluated for state of nuclear maturation. Results show that at 0 h, COCs from Class 3 have fewer oocytes at the GV stage than COCs from Class 1 and Class 2 (respectively 69.3+/-3.2 vs 88.8+/-3.4% and 86.9% GV+/-4.3% SEM; P < 0.05). After 4 h of incubation, all COCs classes show a significant decrease in the number of COCs at the GV stage. The COCs maintained in meiotic arrest and then incubated for 4 h resume meiosis faster than COCs incubated in cycloheximide and theca cell-free medium (19.4+/-2.5, 33.3+/-7.3 and 59.9+/-6.5% GV SEM, respectively). The COCs of Class 3 have fewer oocytes at the GV stage at the beginning of incubation than all other classes. The number of COCs at the GV stage after 4 h of incubation in cycloheximide and theca cell-free medium is not significantly different than those COCs incubated in the presence of theca cell monolayers for 24 h (58.8+/-6.5 vs. 56.4+/-6.4% SEM; respectively). Our results indicate that the ability of theca cells to maintain oocytes at the GV stage could be limited to those oocytes that were not committed or primed in vivo to resume maturation as indicated by their faster maturation kinetics.     

Manipulation of chromosome condensation by protein synthesis inhibitors and cyclic AMP during maturation of bovine oocytes

We investigated the effects of cycloheximide on bovine oocyte chromosomes during meiotic maturation in vitro. Bovine oocytes at Metaphase I (MI) of the meiotic maturation were treated with 10 mug/ml cycloheximide alone or in addition to 5 mM dibutyrylcAMP (dbcAMP) plus 1 mM isobutylmetylxantine (IBMX). A maturation period of 15 to 18 h followed by 12-h treatment with cycloheximide appeared to be most efficient to induce interphase (86% with 16 h maturation). About 60% of oocytes returned to a metaphase state 12 h after the oocytes were transferred to cycloheximide-free medium. In contrast, up to 73% of cycloheximide-treated oocytes at 17 h of maturation remained in interphase if dbcAMP plus IBMX was included in the cycloheximide-free medium. This shows that dbcAMP plus IBMX can inhibit the development of conditions in the oocytes that are required for the transition to metaphase. The chromosome decondensation induced by protein synthesis inhibition at Metaphase I is reversible. This study shows that transition to interphase in bovine oocyte depends on the stage of maturation of oocytes and is sensitive to cAMP levels.     

In vitro inhibition of oocyte nuclear maturation in the bovine

Bovine follicular oocytes (N = 5991) were exposed to an analog of cyclic adenosine 3',5'-monophosphate (cAMP), dibutyryl cyclic AMP (db-cAMP) (2.5, 5, and 10 mM), the phosphodiesterase inhibitor isobutyl methyl xanthine (IBMX, 0.2 mM), or the purine, hypoxanthine (0.5, 1.0, 2.0 mM), and the nucleoside, adenosine (0.05, 0.1, 0.2 mM), for 6 or 21 h to assess their effects on oocyte nuclear maturation. Potential effects of bovine follicular fluid (BFF) were also evaluated after different preculture washing procedures. In a separate experiment, denuded oocytes were used to study the effect of cumulus removal on meiotic inhibition. Db-cAMP decreased the frequency of germinal vesicle breakdown (GVBD) at 6 h (88% for control and 51%, 45%, and 32% for 2.5, 5, and 10 mM concentrations, respectively). IBMX had a comparable effect with only 41% of the oocytes resuming meiosis. Hypoxanthine and adenosine alone or in combinations also decreased the number of oocytes undergoing GVBD at 6 h. Only 22% GVBD occurred when the combined highest concentration of both substances was used compared to 88% in controls. If oocytes were incubated in 50% BFF after a wash in control medium during processing, 56% would resume meiosis at 6 h vs. 35% if the washing procedure included inhibitors (db-cAMP + IBMX). Total BFF (100%) during washing and maturation prevented 72% of the oocytes from resuming meiosis. Db-cAMP and IMBX combined or BFF also inhibited meiotic resumption of denuded oocytes. At 21 h, the inhibitory effects were less pronounced, with most oocytes only delayed in completing the first reduction division.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combined use of dbcAMP and IBMX minimizes the damage induced by a long‐term artificial meiotic arrest in mouse germinal vesicle oocytes

Phosphodiesterase (PDE)‐mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3‐isobutyl‐1‐methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV‐stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double‐strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 μM dbcAMP and 10.0 μM IBMX efficiently inhibited meiotic resumption in GV‐stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.     

山羊卵母细胞的减数分裂进程

The meiotic progression of goat oocytes from follicles of different diameters was investigated in this study. The results were summarized as follows: (1) The in vitro meiotic maturation capacity was different among oocytes from follicles of different diameters. And thus oocytes from < or = 0.5 mm follicles were unable to resume meiosis; oocytes from 0.8-1.2 mm follicles were capable to resume meiosis, but could develop only to MI stage (60% at 24 h); oocytes from 1.5-5 mm follicles had acquired full-meiotic maturation capacity and 91% of them developed to M II stage at 24 h of culture. (2) The percentage of oocytes with intact-germinal vesicles from 1.5-5 mm follicles decreased significantly during 2-8 h of in vitro maturation and the decrease was even more rapid during 4-6 h of culture (from 60% to 19%, p < 0.0005). The percentage of oocytes at M I-stage increased from 24% to 61% during 6-12 h of in vitro maturation, and it then decreased. By 24 h of culture, only 2% oocytes remained at M I-stage. Twenty one percent of the oocytes in this group developed to M II-stage at 16 h of culture, and by 24 h of culture, 91% were at M II-stage. (3) Statistic analysis of the meiotic progression (the duration of each cell cycle stage) of oocytes from 1.5-5 mm follicles showed that GV stage lasted from 0 to 3 h of culture, prometaphase-I stage was from 3.0 to 7.0 h, metaphase-I stage was from 7.0 to 14.6 h, anaphase-I/telophase-I was from 14.6 to 18.4 h and metaphase-II stage lasted from 18.4 to 24 h. (4) Whether the oocytes capable of GVBD and entrance of M I developed to M II, the timing of meiotic progression prior to M I was similar. In summary, our results provided necessary data for studies on the mechanisms and control of meiosis in mammalian oocytes.     

Utility of rapidly matured oocytes as recipients for production of cloned embryos from somatic cells in the pig

The present study was conducted to examine the utility of rapidly matured oocytes as recipients for production of porcine embryos reconstituted with adult skin fibroblasts and whether arrest of meiotic resumption of recipient oocytes at the germinal vesicle (GV) stage by dibutyryl cyclic AMP (dbcAMP) improves in vitro developmental rates after reconstruction. At 24 h of maturation in the medium, 36.3% of oocytes reached the metaphase II (MII) stage. At 30 h of maturation, the percentage (71.4%) of MII oocytes did not significantly differ from that (78.0%) at 42 h of maturation. When MII oocytes recovered at 24 h of maturation were used as recipients, 22/156 (14.1%) cloned embryos developing to the blastocyst stage was significantly (P < 0.05) higher than those of embryos reconstituted with oocytes collected at 30 h (5/168; 3.0%) and 42 h (13/217; 6.0%) of maturation. Culture of oocytes in medium containing 1 mM dbcAMP for 20 h maintained 72.9% in the GV stage, whereas only 15.0% of nontreated oocytes were in the GV stage (P < 0.05). The effect of dbcAMP was reversible. However, the treatment of recipient oocytes with dbcAMP did not affect the development of reconstructed embryos when compared with nontreated oocytes. These results indicate that rapidly matured oocytes are superior in their ability to support development of porcine reconstructed embryos; however, arrest of meiotic resumption of recipient oocytes at the GV stage by dbcAMP does not improve reconstructed embryo developmental rates.     

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes

Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199 + 10 ng/ml epidermal growth factor supplemented with 25 microM beta-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM + dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM + roscovitine (12.5, 25, or 50 microM) for 24 hr. Although approximately 60% of oocytes cultured in 25 microM roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM + 25 microM roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P < 0.05) with approximately 20-30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro.     

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.     

Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes

We investigated effects of invasive adenylate cyclase (iAC), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) on porcine oocyte in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development. Porcine oocytes were collected in Hepes-buffered NCSU-37 supplemented with or without 0.1 microg/ml iAC and 0.5 mM IBMX. IVM was performed in a modified NCSU-37 supplemented with or without 1 mM dbcAMP for 22 h and then without dbcAMP for an additional 24 h. After IVF, oocytes were cultured in vitro for 6 days. After 12 h of IVM, no difference in nuclear status was observed irrespective of supplementation with these chemicals during collection and IVM. At 22 h, most (95%) of the oocytes cultured with dbcAMP remained at the germinal vesicle (GV) stage, whereas 44.3% of the oocytes cultured without dbcAMP underwent GV breakdown. At 36 h, oocytes cultured with dbcAMP had progressed to prometaphase I or metaphase I (MI) (32.6% and 49.3%, respectively), whereas non-treated oocytes had progressed further to anaphase I, telophase I or metaphase II (MII) (13.6%, 14.3% and 38.0%, respectively). At 46 h, the rate of matured oocytes at MII was higher in oocytes cultured with dbcAMP (81%) than without dbcAMP (57%), while the proportion of oocytes arrested at MI was lower when cultured with dbcAMP (15%) than without dbcAMP (31%). The rate of monospermic fertilisation was higher when oocytes were cultured with dbcAMP (21%) than without dbcAMP (9%), with no difference in total penetration rates (58% and 52%, respectively). The blastocyst rate was higher in oocytes cultured with dbcAMP (32%) than without dbcAMP (19%). These results suggest that a change in intracellular level of cAMP during oocyte collection does not affect maturational and developmental competence of porcine oocytes and that synchronisation of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to MII transition and results in high developmental competence by monospermic fertilisation.     

Effects of roscovitine on the nuclear and cytoskeletal components of calf oocytes and their subsequent development

Roscovitine, a potent inhibitor of M-phase promoting factor kinase activity, was used to maintain calf oocytes at the germinal vesicle stage for a 24h culture period. Cumulus-oocyte complexes were first prematured for 24h in the presence of different levels of roscovitine (12.5, 25, 50 and 100 microM). Roscovitine was shown to block germinal vesicle breakdown in calf oocytes in a concentration dependent manner. Significantly greater inhibitory effect was observed at 50 and 100 microM with 64.6% and 63.2% oocytes being blocked in the germinal vesicle stage when compared to the control (0.0%) and the 12.5 microM (2.9%) and 25 microM (18.8%) groups. However, this inhibitory effect of roscovitine was fully reversible since a substantial number of the oocytes resumed meiosis and reached the metaphase II stage after a further 24h of culture in a permissive medium. Cleavage rates and blastocyst yields were not significantly different for oocytes cultured under 50 microM roscovitine inhibition compared to oocytes not subjected to prematuration culture (rates of 76.7% cleavage and 8.7% blastocysts for control oocytes compared to 69.8% and 6.3%, respectively, for oocytes pretreated with 50 microM roscovitine). The morphology of the meiotic spindle was typical of metaphase II in 75.8% and 82.1% of the oocytes reaching the metaphase II stage after pretreatment with 50 microM roscovitine compared to control, respectively. A normal distribution of actin filaments was observed in 97.0% and 98.2% of oocytes exposed to 50 microM roscovitine compared to control, respectively. These results demonstrate the feasibility of maintaining calf oocytes in artificial meiotic arrest without compromising their subsequent developmental competence.     

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.