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.     

Bovine oocytes treated prior to in vitro maturation with a combination of butyrolactone I and roscovitine at low doses maintain a normal developmental capacity.

Butyrolactone I (BL-I) and Roscovitine (ROS), two specific and potent inhibitors of M-phase promoting factor (MPF) kinase activity, were used to block germinal vesicle breakdown (GVBD) of cattle oocytes. A concentration 6.25 microM BL-I and 12.5 microM ROS blocked over 93.3 +/- 2.5% of oocytes in germinal vesicle (GV) stage during a 24-hr culture period. Following a second 24-hr culture step in maturation medium (IVM) almost all (91.5 +/- 3.0%) inhibited oocytes resumed meiosis and reached the metaphase II (MII) stage. The MII kinetics was different for inhibited and control oocytes. Fifty percent MII was reached at 13-14 hr in BL-I + ROS treated oocytes, compared to 18 hr in control oocytes. Therefore, control oocytes were fertilised (IVF) after 22 hr IVM and inhibited oocytes after 16 or 22 hr IVM. After IVF, percentage of grade 1 freezable embryos on day 7 (D + 7) as well as percentage of blastocyst formation on D + 8 in the group of BL-I + ROS treated oocytes fertilised after 16 hr IVM were higher (P < 0.05) compared with the other experimental group fertilised after 22 hr IVM but not different in comparison with the control. Survival to freezing and thawing of grade 1 embryos frozen on D + 7 was employed as viability criteria and was similar in all groups. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes determines a reversible meiotic block, without compromising their subsequent developmental competence.     

Effects of cell cycle dependent kinases inhibitor on nuclear and cytoplasmic maturation of porcine oocytes

The aims of this study were to assess the effectiveness of roscovitine, a potent inhibitor of cell cyclin kinases, to prevent meiotic resumption in porcine oocytes, and to test the subsequent fertilisability and developmental competence of these oocytes. Roscovitine blocked porcine oocytes at the GV stage during 22-44 hr of culture. This effect was dose-dependent, and a concentration of 25 microM was sufficient to prevent meiotic resumption in 92+/-5% of the oocytes after 22 hr in the presence of EGF and FSH. Cumulus expansion was also inhibited under these conditions. The histone H1 kinase activity in oocytes was inhibited in a dose-dependent way, and maintained at a basal level with 25 microM of roscovitine. Synthesis of proteins of 29, 47 and 79 kDa, normally synthesized during maturation, was inhibited too. All these effects were fully reversible. However, the kinetics of maturation were accelerated after roscovitine removal, and the acceleration was more pronounced after 44 hr of inhibition than after 22 hr. Fertilization of oocytes blocked for 22 hr before a 44 hr maturation was decreased compared to control, but was not different from that of oocytes matured for 66 hr. The developmental competence was decreased for the oocytes cultured for 66 hr, including or not an inhibition period, but it was less reduced for oocytes maintained under inhibition for 22 hr. Roscovitine may thus protect oocytes against the aging mechanisms responsible for developmental competence loss, but not against loss of fertilisability. In conclusion, roscovitine provides a useful tool to study the morphological and biochemical basis of porcine oocyte terminal differentiation.     

Vitrification of calf oocytes: effects of maturation stage and prematuration treatment on the nuclear and cytoskeletal components of oocytes and their subsequent development

This study was designed to establish the effects of the meiotic stage of bovine oocytes and of a prematuration treatment with roscovitine (ROS) on their resistance to cryopreservation. Oocytes from prepubertal calves at the stages of germinal vesicle breakdown (GVBD) or at metaphase II (MII) were vitrified by the open pulled straw (OPS) method. In another experiment, oocytes were kept under meiotic arrest with 50 microM ROS for 24 hr and vitrified at the GVBD stage. After warming, some oocyte samples were fixed, stained using specific fluorescent probes and examined under a confocal microscope. The remaining oocytes were fertilized, and cleavage and blastocyst rates recorded. Significantly lower cleavage rates were obtained for the vitrified GVBD and MII oocytes (9.9% and 12.6%, respectively) compared to control oocytes (73.9%). Significantly worse results in terms of cleavage rates were obtained when GVBD calf oocytes were exposed to cryoprotectants (CPAs: ethylene glycol plus dimethyl sulfoxide, DMSO) (13.1%) or vitrified (1.6%) after a prematuration treatment with ROS, when compared to untreated control oocytes (68.7%) or ROS-control oocytes (56.6%). None of the vitrification procedures yielded blastocysts, irrespective of the initial meiotic stage or previous prematuration treatment. Compared to the control oocytes, significantly fewer oocytes exhibited normal spindle configuration after being exposed to CPAs or after vitrification of either GVBD or MII calf oocytes. These results indicate that the vitrification protocol has a deleterious effect on the meiotic spindle organization of calf oocytes cryopreserved at both the GVBD and MII stage, which impairs the capacity for further development of the embryos derived from these vitrified oocytes. Prematuration treatment with ROS has no beneficial effect on the outcome of vitrification by the OPS method.     

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.     

Protein synthesis and mRNA storage in cattle oocytes maintained under meiotic block by roscovitine inhibition of MPF activity

Roscovitine, a specific inhibitor of MPF kinase activity, has been shown to block efficiently and reversibly the meiotic resumption of oocytes from different species, including cattle. In view to verify that oocytes maintain germinal vesicle like molecular activities under roscovitine treatment, we compared in the present study the M-phase Promoting Factor (MPF) and Mitogen Activated Protein (MAP) kinase activities; protein synthesis and phosphorylation patterns in oocytes and cumulus cells; and CDK1 and Cyclin B messengers storage under control culture and under roscovitine inhibition. We observed that roscovitine induced a full and reversible inhibition of MPF kinase activity and of the activating phosphorylation of both ERK1/2 MAPK. During in vivo maturation, there was a highly significant increase in the relative mRNA level of both cyclin B1 and CDK1 whereas during in vitro culture, the relative amount of CDK1 messenger was reduced. These messengers may be used as markers for the optimization of in vitro maturation treatment. Roscovitine reversibly prevented this drop in relative quantities of CDK1 messenger. Oocytes cultured in the presence of roscovitine maintained a GV like profile of protein synthesis except that two proteins of 48 and 64 kDa specific of matured oocytes also appeared under roscovitine treatment. However, roscovitine did not prevent most of the modifications of protein phosphorylation pattern observed during maturation. In conclusion, results of this study revealed that the use of roscovitine did not prevent all the events related to maturation of bovine oocytes.     

The effects of roscovitine on cumulus cell apoptosis and the developmental competence of domestic cat oocytes

The developmental competence of cat oocytes matured in vitro is relatively poor when compared with that of in vivo oocytes. The study aimed to investigate the effect of roscovitine on the developmental competence of cat Felis catus oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were classified as Grade I and II to III. Groups of COCs were cultured in 0, 12.5, 25, 50, 100, and 200 μM roscovitine for 24 h and were either fixed to assess the stages of nuclear maturation (Experiment 1) or additionally matured in vitro for 24 h before fixation (Experiment 2). In Experiment 3, cumulus cells from the COCs treated with roscovitine were examined for apoptosis. Experiment 4 examined the developmental competence of cat oocytes after roscovitine treatment and in vitro fertilization in terms of cleavage and morula and blastocyst formation rates. Roscovitine reversibly arrested cat oocytes at an immature stage in a dose-dependent manner. Roscovitine at 12.5 and 25 μM demonstrated less efficiency compared with that of other doses. However, higher doses of roscovitine induced cumulus cell apoptosis and resulted in a high number of degenerated oocytes after in vitro maturation. Roscovitine at 12.5 and 25 μM were therefore used to evaluate their effect on embryo development. Pretreatment with 12.5 and 25 μM roscovitine prior to in vitro maturation decreased the developmental competence of cat oocytes compared with that of non-roscovitine-treated controls. In conclusion, roscovitine reversibly maintained cat oocytes at the germinal vesicle stage without detrimental effect on nuclear maturation. However, it negatively affected cumulus cell viability and developmental competence.     

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.     

Effect of cycloheximide, 6-DMAP,roscovitine and butyrolactone I on resumption of meiosis in porcine oocytes

Improvement of the ability to maintain germinal vesicle stage oocytes in vitro is important for the acquisition of developmental competence. Maintaining oocytes at this stage without damaging their quality would allow synchronization of maturation and homogenization of the oocytes population. More investigations are needed to better understand how the oocyte cell cycle is blocked without consequences to future developmental competence. This study tested the efficacy of pharmacological inhibitors of the G2/M cell cycle transition in keeping porcine oocytes at the germinal vesicle (GV) stage and the reversibility of this inhibition. Porcine cumulus-oocyte complexes (COCs) were thus incubated without any hormones for 24 h in the presence or absence of tested inhibitors: 6-DMAP (protein kinase inhibitor, 2 mM), cycloheximide (protein synthesis inhibitor, 2 microg/ml), roscovitine (cyclin-dependent kinase inhibitor, 50 microM) and butyrolactone I (cyclin-dependent kinase inhibitor, 50 microM). Cumulus-oocyte complexes cultured with any of the inhibitors were significantly blocked at the GV stage. The inhibitory effect varied according to the products, with cycloheximide being the most efficient. Reversibility of the pharmacological inhibitors was assessed by culturing COCs an additional 24 h in inhibitor-free culture medium. Examination of oocytes revealed that the inhibitory effect was fully reversible. This study suggests that 6-DMAP, cycloheximide, roscovitine and butyrolactone I can be use to block meiotic resumption in porcine oocytes in NCSU culture medium.     

Nuclear and cytoplasmic maturation of sow oocytes are not synchronized by specific meiotic inhibition with roscovitine during in vitro maturation

The effect of roscovitine exposure prior to IVM was studied on cumulus expansion, on changes of cumulus-oocyte contacts and on nuclear and cytoplasmic maturation of sow oocytes. It was hypothesized that delayed nuclear maturation and prolonged contact with cumulus cells allows prolonged cytoplasmic differentiation and therefore improves oocyte developmental potential. Cumulus-oocyte complexes (COCs) were exposed for 22 h or 44 h to 0, 25 or 50 microM of roscovitine and subsequently cultured for 22, 29 or 44 h without roscovitine. COCs were examined for cumulus expansion and oocytes for nuclear status and dynamics of transzonal microfilaments. Oocyte developmental potential was assessed by blastocyst formation after IVF. Fifty muM of roscovitine inhibited cumulus expansion for the first 22 h of culture, and maintained oocytes in meiotic arrest for 44 h. Roscovitine treatment during 22 h prior to culture for 44 h without roscovitine did not increase embryo development, but oocytes cultured for 66 h without roscovitine had reduced blastocyst formation. Oocytes cultured for 29 h after roscovitine exposure showed reduced blastocyst rates compared with their counterparts cultured for 44 h. Roscovitine treatment during 44 h prior to culture for 22 h or 44 h without roscovitine reduced embryo development. Transzonal microfilaments were reduced after culture with roscovitine, and disappeared during culture without roscovitine. It is concluded that prolonged contact with cumulus cells does not improve oocyte developmental potential. Furthermore, it is suggested that nuclear and cytoplasmic maturation in vitro cannot be seen as two independent processes.     

Interactive effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes

The objective of this article was to study the effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes. Goat oocytes were cultured at different temperatures in medium containing different concentrations of ROS, and at the end of culture, oocytes were either matured or processed for light/confocal microscopy. The matured oocytes were activated chemically or fertilized in vitro for embryo development. Meiotic arrest was successfully maintained for 24 hr with 0, 50, and 200 microM ROS at 5, 20, and 38.5 degrees C, respectively. Following chemical activation, morulae/blastocysts (M/B) rates similar to untreated oocytes were obtained in oocytes that had been inhibited for 24 hr at 5 degrees C without ROS (Protocol 5C) or at 20 degrees C with 50 microM ROS (Protocol 20C) or for 8 hr at 38.5 degrees C with 200 microM ROS (Protocol 8 hr), but no blastulation was observed after oocytes were inhibited at 38.5 degrees C with 200 microM ROS for 24 hr. Following fertilization, however, while M/B rates similar to controls were achieved in oocytes treated with protocols 5C and 20C, few oocytes inhibited with Protocol 8 hr developed into morulae, due to a high incidence of polyspermy. Changes in GV chromatin configuration were not observed after inhibition with Protocol 5C, but were apparent after inhibition with protocols 20C and 8 hr, leading to a precocious germinal vesicle breakdown (GVBD) during subsequent maturation. Cortical granule (CG) migration and the formation of microtubule organizing centers occurred during inhibition and were more obvious in the absence of ROS. Significantly more oocytes inhibited by protocols 5C and 20C than by Protocol 8 hr completed CG migration after maturation. In conclusion, goat oocytes were tolerant to chilling and culture at lower temperatures with less ROS was better than culture at higher temperatures with more ROS for oocyte GVBD inhibition.     

Role of roscovitine and IBMX on kinetics of nuclear and cytoplasmic maturation of bovine oocytes in vitro

The 3-isobutyl-1-methylxanthine (IBMX) is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte, and roscovitine, a purine known to specifically inhibit MPF kinase activity, maintains bovine oocytes at the germinal vesicle (GV) stage. The present study was conducted to analyze whether cytoplasmic maturation (examined by the pattern of cortical granule (CG) distribution) of bovine oocytes is improved during meiotic arrest with IBMX and roscovitine. Oocytes were matured in vitro in a 10% Knockout(SR) supplemented TCM-199 medium (Control) with either 0.5 mM IBMX or 25 microM roscovitine (ROSC). Oocytes were stained with fluorescein isothiocyanate conjugated Lens culinaris agglutinin (FITC-LCA) for CG evaluation and with Hoechst 33342 for nuclear stage assessment. At 16 h of culture, the percentage of oocytes remaining in the GV stage was higher (P < 0.05) in the ROSC group (32.41%) compared with the Control and IBMX groups (8.61% and 9.73%, respectively). At 24 h of culture, progression of meiosis to M II stage was retarded (P < 0.05) in the ROSC group (24.05%) compared to the Control (60.20%), whereas the IBMX group (33.88%) showed no significant difference to the other two groups. At 16 h of maturation, the proportion of oocytes with CG in clusters (immature cytoplasm) was similar between the groups, as was the percentage of peripheral CG (mature) at 24 h of maturation. The results of the present study demonstrated that the meiotic inhibitors IBMX and roscovitine delay the progression of nuclear maturation without affecting cytoplasmic maturation, assessed by the analysis of CG repositioning.     

Bovine oocyte and embryo development following meiotic inhibition with butyrolactone I

In this study we have shown that butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, inhibits meiotic resumption in bovine oocytes by blocking germinal vesicle breakdown in a dose-dependent manner. A concentration 100 microM blocked over 60% of oocytes, while 150 microM inhibited almost all oocytes compared to the control in which over 80% resumed meiosis. Following a second 24 hr culture under conditions permissive to normal maturation, almost all (95%) of blocked oocytes resumed meiosis and progressed to metaphase II. In terms of developmental competence, oocytes maintained in meiotic arrest for 24 hr with 100 microM exhibited a similar capacity to develop to the blastocyst stage as nonblocked control oocytes following maturation, fertilization, and culture in vitro. Cryopreservation was employed as a tool to detect differences in the oocyte viability between blocked and control oocytes. Cleavage of oocytes was significantly reduced following vitrification and activation both in BL-I treated (40.2% vs. 71.9%, P < 0.05) and the control groups (45.6% vs. 81.7%, P < 0.05). However, BL-I treated oocytes were less likely to develop into blastocysts following vitrification (20.0% from vitrified vs 42.5% from nonvitrified cleaved oocytes, P < 0.05, based on cleaved oocytes) compared to nontreated oocytes (34.0% from vitrified vs. 42. 9% from nonvitrified oocytes, P < 0.05). These results demonstrate the feasibility of maintaining bovine oocytes in artificial meiotic arrest without compromising their subsequent developmental competence and may represent a tool for improving the development of less competent oocytes.     

Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes.

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 microM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 microM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to > or = 22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.     

Embryonic and foetal development of bovine oocytes treated with a combination of butyrolactone I and roscovitine in an enriched medium prior to IVM and IVF

Cattle oocytes were maintained at germinal vesicles (GV) stage for 24 hr using a combination of two specific and potent inhibitors of M-phase promoting factor (MPF) kinase activity, butyrolactone I (BL-I) and roscovitine (ROS). The media used for inhibition were (a) TCM-199 only and (b) TCM-199 supplemented with serum, hormones and growth factors. The effective doses of inhibitors were 6.25 microM BL-I and 12.5 microM ROS in medium (a) and 50 microM BL-I and 12.5 microM ROS in medium (b). After inhibition, about 90% of the oocytes resumed meiosis and reached the metaphase II (MII) stage during 24 hr of maturation. Following fertilisation the percentage of cleavage (D +2), compacted morula (D +6), blastocysts on D +7 and D +8 and the survival to freezing and thawing of grade 1 embryos frozen on D +7 were not different between the experimental treated groups and the control. In order to evaluate early foetal development, two groups of five grade 1 D +7 blastocysts derived from treated oocytes and two groups of five control embryos were transferred nonsurgically in four synchronised recipient heifers. On D +27, the recipients were slaughtered and the foetuses were recovered. In both groups, six foetuses developed out of the 10 embryos transferred. In conclusion, several supplements can be added to the prematuration medium of bovine oocytes without reducing the quality of inhibition but also without improving their subsequent developmental competence versus treated oocytes in TCM-199 only and versus untreated control. Furthermore, the prematuration step used in this study does not interfere with normal foetal development during the first stages of organogenesis.     

Blastocyst development in equine oocytes with low meiotic competence after suppression of meiosis with roscovitine prior to in vitro maturation

This study was conducted to evaluate the in vitro development of equine oocytes with compact cumuli that had been subjected to a period of meiotic suppression with roscovitine before in vitro maturation. In experiment 1, oocytes were recovered from slaughterhouse-derived ovaries and held in M199 + 10% fetal bovine serum containing 66 microM roscovitine with or without an overlay of mineral oil in 5% CO2 in air at 38.2 degrees C for 16-18 or 24 h. No oocytes treated with roscovitine in the absence of an oil overlay for 16-18 h were maturing, compared with 2-4% of oocytes in other treatments. In experiment 2, oocytes were either fixed immediately after recovery, or were cultured for 18 h in the presence or absence of roscovitine. Oocytes cultured in the absence of roscovitine had a significantly higher rate of meiotic resumption (18%) than was found in the other two treatments (0). In experiment 3, oocytes were matured immediately or after 16-18 h culture with roscovitine. Maturation rates were similar between oocytes previously treated with roscovitine (22%) and control oocytes (19%). Mature oocytes were fertilized by intracytoplasmic sperm injection and then cultured, with or without oviductal epithelial cells, for 7.5 days. There was no significant effect of roscovitine treatment on blastocyst development. Development to blastocyst of roscovitine-treated oocytes in DMEM/F-12 + co-culture (37%) was significantly higher than that of control oocytes in DMEM/F-12 without co-culture (14%). These data indicate that equine oocytes with compact cumuli can be held in roscovitine before maturation without any harmful effect on blastocyst formation.     

Holding immature equine oocytes in the absence of meiotic inhibitors: effect on germinal vesicle chromatin and blastocyst development after intracytoplasmic sperm injection

Holding immature oocytes before the onset of maturation simplifies oocyte transport and aids in scheduling later manipulations. We report here a method for holding equine oocytes in the absence of meiotic inhibitors. In Experiment 1, immature oocytes with expanded cumuli were cultured at 38.2 degrees C in medium containing cycloheximide, or were held at room-temperature in M199 with Hanks' salts, for 16-18 h before maturation. Control oocytes were matured immediately after recovery. Oocytes were fertilized by intracytoplasmic sperm injection and cultured for 4d. Embryo development was not different among treatments. In Experiment 2, oocytes were treated as in Experiment 1, but embryos were cultured for 7.5d. Blastocyst development was significantly lower in the cycloheximide-treated group than in controls (7% versus 30%) with the room-temperature group intermediate (16%). In Experiment 3, oocytes were cultured at 38.2 degrees C in medium containing roscovitine, or were held at room temperature in sealed glass vials in a mixture of 40% M199 with Earle's salts, 40% M199 with Hanks' salts, and 20% FBS (EH treatment) for 16-18 h, before maturation, sperm injection, and embryo culture for 7.5d. Blastocyst development of oocytes in the EH treatment was significantly higher than that for roscovitine-treated oocytes (34% versus 12%), but not significantly different from that for controls (25%). Oocytes in the EH treatment did not mature during holding (70% germinal vesicle stage after 18 h holding). Whereas culture with cycloheximide or roscovitine of equine oocytes with expanded cumuli reduced subsequent blastocyst formation, these oocytes could be held in a modified M199 at room temperature overnight without adverse affecting meiotic or developmental competence.     

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.     

High developmental competence of pig oocytes after meiotic inhibition with a specific M-phase promoting factor kinase inhibitor,butyrolactone I

Butyrolactone I specifically inhibits M-phase promoting factor activation and prevents the resumption of meiosis. These experiments were conducted to examine effects of butyrolactone I on pig oocytes in a serum-free maturation system. The first experiment was conducted to determine the effect of butyrolactone I (0-100 microM) on nuclear maturation. At concentrations of > or =12.5 microM, germinal vesicle breakdown was prevented in >90% of the oocytes after 24 h of culture. In the second experiment, the kinetics of in vitro maturation of butyrolactone I-treated oocytes was investigated. Oocytes were treated with 0 or 12.5 microM butyrolactone I and FSH for 20 h and then cultured with LH in the absence of butyrolactone I for another 24 h. Fewer butyrolactone I-treated oocytes reached MII stage at 36 h compared with controls (5.8% vs. 62.4%, P < 0.01). However, by 44 h, 83.4% of butyrolactone I-treated oocytes reached MII compared with 88.6% of controls. In the third experiment, butyrolactone I-treated oocytes were fertilized and cultured in vitro. No differences (P > 0.05) were found between controls and treated groups in cleavage rate, blastocyst rate, or mean number of cells per blastocyst. Effects of butyrolactone I on mitogen-activated protein kinase activation and localization of microfilaments and active mitochondria were examined by Western blot analysis and laser scanning confocal microscopy, respectively. The results suggested that although butyrolactone I reversibly inhibited germinal vesicle breakdown and mitogen-activated protein kinase activation, it did not affect mitochondrial and microfilament dynamics. Butyrolactone I is a potent inhibitor of nuclear maturation of porcine oocytes, and the inhibition is fully reversible.     

Effect of roscovitine on nuclear maturation, MPF and MAP kinase activity and embryo development of prepubertal goat oocytes

The low number of embryos obtained from IVM-IVF-IVC of prepubertal goat oocytes could be due to an incomplete cytoplasmic maturation. Roscovitine (ROS) inhibits MPF and MAP kinase activity and maintains the oocyte at Germinal Vesicle (GV) stage. The aim of this study was to determine if meiotic activity is arrested in prepubertal goat oocytes cultured with 0, 12.5, 25, 50 and 100 microM of ROS for 24 h. A group of oocytes from adult goats was cultured with 25 microM of ROS to compare the effect of ROS on prepubertal and adult goat oocytes. A sample of oocytes was stained to evaluate the nuclear stage at oocyte collection time and after ROS incubation. IVM-oocytes not exposed to ROS formed the control group. Prepubertal goat IVM-oocytes were inseminated and cultured for 8 days. The percentage of oocytes at GV stage, after exposition to ROS was significantly higher in adult goat oocytes (64.5%) than in prepubertal goat oocytes. No differences were found among 25, 50 and 100 microM ROS concentrations (29, 23 and 26%, oocytes at GV stage, respectively). After 8 days of culture, no differences in total embryos were observed between control oocytes and oocytes treated with 12.5 and 25 microM (45.2, 36.1 and 39.4%, respectively), however the percentage of blastocysts was higher in the control group. Western blot for the MAPK and p34(cdc2) showed that both enzymes were active in prepubertal goat oocytes after 24h of ROS exposition. In conclusion, a low percentage of prepubertal goat oocytes reached GV stage after ROS incubation; possibly because most of them had reinitiated the meiosis inside the follicle. ROS did not affect fertilization or total embryos but ROS showed a negative effect on blastocyst development.