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Regular Article |
a Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211
b Monsanto, St. Louis, Missouri 63198
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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12.5 µM, 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 µM 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.
early development, gamete biology, kinases, meiosis, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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M-phase promoting factor (MPF), which consists of 2 subunits p34cdc2 and cyclin B, is a critical factor that regulates the resumption of meiosis in oocytes [3]. MPF activation requires new protein synthesis [4], phosphorylation of p34cdc2 at T14, Y15, and T161, and subsequent dephosphorylation of p34cdc2 at T14 and Y15 [5]. Interference in any of those processes will inhibit MPF activation, prevent resumption of meiosis, and hold the oocyte at the GV stage. For example, elevation of intracellular cAMP levels by treatment with dbcAMP [1] or hypoxanthine [6], an inhibitor of cAMP-phosphodiesterase, will keep MPF in a hyperphosphorylated inactive state. Nonspecific inhibition of protein synthesis by cycloheximide has been shown to prevent activation of MPF [7, 8]. Nonspecific inhibition of protein phosphorylation with 6-dimethylaminopurine treatment will also inhibit MPF activation [9]. Although these methods effectively inhibit MPF activation, their nonspecific mechanism of action is either harmful to subsequent embryonic developmental competence [7–9] or low in efficiency [1]. Recently, butyrolactone I (BL-I) and roscovitine, purine derivatives designed to specifically inhibit MPF activation by competing with ATP binding to p34cdc2 thus preventing its dephosphorylation [10–12], have been successfully used to maintain bovine [13–15] and porcine [16, 17] oocytes at the GV stage. The advantage of this method is that GVBD can be prevented by specifically inhibiting MPF activation without suppressing protein synthesis, phosphorylation, or MPF accumulation, all of which are required for complete oocyte maturation.
Recently, bovine oocytes were maintained at the GV stage for 24 h by treatment with BL-I or roscovitine without compromising their subsequent developmental competence [13–15]. BL-I effectively and reversibly prevented resumption of meiosis in porcine and bovine oocytes [16]. Although BL-I can interrupt meiotic progression of pig oocytes, its effects on cytoskeletal architecture, cellular function, and subsequent developmental potential are unknown. In the current study, experiments were conducted on pig oocytes to examine the ability of BL-I to reversibly inhibit GVBD. The time course of nuclear maturation and subsequent developmental potential were determined following removal of BL-I. To investigate potential beneficial or detrimental effects of delaying meiotic progression on cellular function, the distributions of microfilaments and active mitochondria were examined by laser scanning confocal microscopy. Furthermore, the activation of mitogen-activated protein (MAP) kinase in oocytes treated with BL-I was also examined to assess its effects on cytoplasmic maturation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Butyrolactone I (Biomol, Plymouth Meeting, PA) was prepared as a 50 mM stock solution in dimethyl sulfoxide. All other chemicals were from Sigma (St. Louis, MO) unless otherwise indicated.
Oocyte Collection and IVM
Immature oocytes were aspirated from 2- to 5-mm follicles of ovaries obtained from a local abattoir. Oocytes with uniform ooplasm and compact cumulus were collected and washed in Hepes-buffered Tyrode medium containing 0.1% polyvinyl alcohol (PVA) (w/v). After washing 3 times with basic maturation medium (TCM 199; Gibco, Grand Island, NY) supplemented with 0.1% PVA, 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng/ml epidermal growth factor (EGF), 50 IU/ml penicillin K salt, and 50 IU/ml streptomycin sulfate [18], groups of 50 oocytes were transferred into individual wells of a four-well Nunclon dish with 0.5 ml basic culture medium with 0.5 µg/ml FSH and were cultured for 20 h at 38.5°C with or without BL-I. The oocytes were then washed 6 times and cultured for up to 24 h in basic culture medium with 0.5 µg/ml LH. After culture, oocytes were stripped of cumulus cells by pipetting with 0.1% (w/v) hyaluronidase.
In Vitro Fertilization and Embryo Culture
Cumulus-free oocytes were washed 3 times with fertilization medium, and 30–35 oocytes were transferred into 50-µl droplets of fertilization medium covered with mineral oil (Fisher Scientific, Pittsburgh, PA) that had been equilibrated for 40 h at 38.5°C in 5% CO2 in air. The dishes were kept in a CO2 incubator until sperm were added for insemination. The fertilization medium was a modified Tris-buffered medium consisting of 110 mM NaCl, 0.47 mM KCl, 7.5 mM CaCl2, 0.5 mM sodium pyruvate, 10 mM glucose, 20 mM Tris, 2 mM caffeine, and 2 mg/ml BSA [19]. For in vitro fertilization (IVF), one 0.1-ml frozen semen pellet was thawed at 39°C in 10 ml DPBS (Gibco) containing 1 mg/ml BSA, 50 IU/ml penicillin K salt, and 50 IU/ml streptomycin sulfate. After washing 2 times by centrifugation (1900 x g, 4 min), cryopreserved ejaculated spermatozoa were resuspended with fertilization medium to a concentration of 6 x 105 cells/ml, and 50 µl of the sperm sample was added to the fertilization droplets containing the oocytes. Six hours postinsemination (p.i.), oocytes were washed 3 times and cultured in 0.5 ml culture medium (NCSU 23 containing 4 mg/ml BSA) [20] in four-well Nunclon dishes (Nunc, Roskilde, Denmark). Some oocytes were fixed 12 h p.i. in acetic ethanol (25%, v/v) to examine fertilization status. Cleavage was examined at 48 h p.i., and blastocyst formation was examined at 144 h p.i. Only embryos with blastomeres of equal size were counted in cleavage rate. Apparent blastocysts were stained with Hoechst 33342 and mounted on slides for counting of cell nuclei. Embryos with <15 nuclei were not defined as blastocysts in these experiments.
Laser Scanning Confocal Microscopy of Mitochondria and Microfilaments
For mitochondrial examination, denuded oocytes were stained with 0.5 µM MitoTracker Green FM (Molecular Probes, Eugene, OR) for 30 min in fresh basic maturation medium at 38.5°C under 5% CO2 in air. After washing 3 times in basic maturation medium under the above conditions, oocytes were fixed in 3.7% (w/v) paraformaldehyde in PBS for 2 h at room temperature. The oocytes were then washed twice in PBS, stained with 10 µg/ml propidium iodide (PI) for 2 h, and mounted on slides as previously described [21].
For microfilament staining, denuded oocytes were fixed in 3.7% paraformaldehyde in PBS (pH 7.4) for 2 h at room temperature. After fixation, embryos were treated with 1% (v/v) Triton X-100 in PBS for 6 h at room temperature or overnight at 4°C, washed twice in PBS, and cultured in a blocking solution (PBS containing 2% BSA w/v and 150 mM glycine) for 30 min at room temperature. After being washed for another hour in PBS, the embryos were stained with 10 IU/ml fluorescein isothiocyanate (FITC)-phalloidin for 1 h at 38.5°C in PBS with Tween-20 (0.1%, v/v). After washing in PBS with Tween-20 (0.1%, v/v) for 2 h at room temperature, the oocytes were stained with 10 µg/ml PI for 2 h and mounted on slides [22].
Confocal microscopy was performed by using a MRC-600 confocal laser scanning imaging system (Bio-Rad, Richmond, CA) equipped with krypton/argon laser guns and 60x objectives. MitoTracker and FITC were excited at 488 nm, and excitation of PI occurred at 568 nm. The image was obtained by repeated Kalman laser scanning method (8 times during 8 sec) with diminished background noise.
MAP Kinase Phosphorylation Assay
To determine whether treatment with BL-I inhibited activation of MAP kinase, proteins from a total of 30 oocytes per treatment were extracted with double-strength electrophoresis buffer. After boiling for 3 min and centrifuging for 3 min at 14 000 x g, the lysates were kept frozen at -80°C until use. Proteins were separated on a 10% SDS-polyacrylamide gel for 1 h at 188 V and then transferred onto Immobilon-P transfer membranes (Millipore Co., Bedford, MA) for 1 h at 200 mA [23]. Membranes were immersed in methanol for 1 min, dried overnight at room temperature, and then incubated for 2 h at room temperature with polyclonal rabbit anti-phosphorylated MAP kinase antibody (New England Biolabs, Beverly, MA) diluted 1:600 in PBS containing 5% skim milk (pH 7.4). This antibody recognizes both isoforms of phosphorylated MAP kinase, pERK1 and pERK2. After 2 washes of 5 min each in PBS containing 0.01% Tween-20 (PBS-T), membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:2000. Membranes were then washed twice in PBS-T for 5 min each time and processed using the ECL detection system (Amersham International, Little Chalfont, Buckinghamshire, U.K.) [24].
Experiment 1: Effect of BL-I on Nuclear Maturationof Pig Oocytes
Oocytes (n = 942) were cultured in the basic maturation medium containing 0.5 µg/ml FSH supplemented with 0, 6.25, 12.5, 25, 50, or 100 µM BL-I for 24 h, fixed in 25% (v/v) acetic alcohol for 48 h, and stained with 1% orcein to determine the effective dose of BL-I in preventing GVBD. Nuclear status of oocytes was categorized as GV (GVI–GVIV), MI (diakinesis and metaphase I), MII (anaphase I, telophase I, and metaphase II), and degenerated. In a second series of experiments, oocytes were treated with 12.5 µM BL-I for 24 h, fixed to examine meiotic maturation, and compared with freshly aspirated oocytes. The GV stages were classified as described by Motlík and Fulka [2].
Experiment 2: Time Course of Nuclear Maturation Following Treatment with BL-I
After 20 h of culture with 12.5 µM BL-I in basic medium with 0.5 µg/ml FSH, oocytes were washed 6 times and cultured in basic maturation medium with 0.5 µg/ml LH for up to 24 h. Oocytes were fixed with ethanol-acetic acid, and their nuclear morphology was examined after 0, 8, 16, and 24 h of culture in the LH medium (total culture times: 20, 28, 36, and 44 h). Control oocytes without BL-I treatment were fixed at the same time points.
Experiment 3: Developmental Potential of BL-I-Treated Oocytes
After 20 h of culture with or without 12.5 µM BL-I in basic medium with 0.5 µg/ml FSH, oocytes were washed 6 times and cultured in basic maturation medium with 0.5 µg/ml LH for an additional 24 h and then fertilized in vitro and cultured to examine fertilization parameters (penetration rate, polyspermy rate, number of sperm per penetrated oocyte, and decondensation rate of penetrated sperm), cleavage rate, and blastocyst formation. The blastocysts were also stained with 2 µg/ml Hoechst 33342, and number of nuclei per blastocyst was counted under an ultraviolet microscope.
Experiment 4: Effects of BL-I Treatment on Activationof MAP Kinase and Distribution of Active Mitochondria and Microfilaments of Pig Oocytes
Oocytes were cultured with or without 12.5 µM BL-I in basic medium with 0.5 µg/ml FSH for 24 h, washed, and cultured in basic maturation medium with 0.5 µg/ml LH for an additional 20 h. After total maturation times of 0, 24, and 44 h, the oocytes were denuded with 0.1% hyaluronidase and mitochondria and microfilaments were examined with a confocal microscope. Some of the oocytes pooled at a total maturation time of 24, 32, and 44 h were used for a MAP kinase phosphorylation assay. The cell lysate from each treatment group (30 oocytes/treatment) was electrophoresed on the same polyacrylamide gel. The result was examined by direct comparison of the intensity of immunoreactivity of the antiphosphorylated MAP kinase antibody in each lane.
Statistical Analysis
Data were analyzed by ANOVA and a Duncan multiple range test using the general linear models in the Statistical Analysis System (SAS Institute, Cary, NC) program to determine treatment differences. All data are expressed as mean ± SEM. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast to the control group, in which only 21.5% of oocytes remained at the GV stage after 24 h of culture, 78.0% of oocytes treated with 6.25 µM BL-I remained at the GV stage. Maximal inhibition of BL-I was observed at concentrations of 12.5 µM, where >90% of the oocytes remained in the GV stage after 24 h of culture (Fig. 1). Therefore, 12.5 µM was determined to be the optimal concentration of BL-I and was used for all subsequent experiments.
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Typical morphology of different GV stages is shown in Figure 2. BL-I inhibited the nuclear maturation process completely at 12.5 µM, as judged by evaluation of nuclear morphology. Comparison of GV stages of oocytes cultured with BL-I for 24 h with those of uncultured, freshly aspirated oocytes (0 h) revealed no differences in percentage of oocytes at each meiotic stage (Table 1). BL-I inhibited nuclear development of porcine oocytes at all stages. No difference was observed in the rate of degenerated oocytes in all 3 groups at the end of BL-I treatment.
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Inhibition of Nuclear Maturation by BL-I Is Fully Reversible
In the second experiment, the reversibility of the progression of the nuclear maturation was examined after removal of BL-I (Fig. 3). To minimize variation of experimental conditions by inseminating the oocytes in treatment and control groups with the same pellet of sperm, the oocytes were treated with BL-I for 20 h instead of 24 h as in the previous experiment.
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After removal of the inhibitor, 66% of the oocytes reached diakinesis or MI in 8 h. Over the next 8 h, only 5.8% developed to MII, whereas 61.4% of oocytes in the control group had reached MII by that time. Twenty-four hours after the removal of BL-I, 83.4% of oocytes had completed nuclear maturation with a 4.8% increase in degenerated oocytes (P = 0.02) compared with 88.6% in the control group. This 5.2% difference was significant (P = 0.03).
Developmental Potential of Porcine OocytesIs Not Affected by BL-I Treatment
BL-I treatment had no effect on fertilization rate, polyspermy rate, or average number of sperm per penetrated oocyte (Table 2). In this experiment, all of the oocytes that had condensed sperm heads also had at least 2 pronuclei, with only one exception. Therefore, male pronucleus formation rate was not calculated for penetrated oocytes. To determine the ability of oocytes to decondense the penetrated sperm, an important evaluation parameter of cytoplasmic maturation, the rate (percentage) of pronuclear formation for penetrated sperm was calculated (Table 2). No significant difference was detected between treated and control groups (P > 0.05). BL-I treatment prior to IVM also had no effect on cleavage rates or blastocyst formation rates following IVF (Table 3). However, there was a trend toward increased number of cells per blastocyst when oocytes were treated with 12.5 µM BL-I for 20 h (P = 0.11). A typical view of Day 6 blastocysts is shown in Figure 4.
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Cytoplasmic Maturation in Relation to Nuclear Progression in BL-I-Treated Oocytes
Confocal microscopy indicated that BL-I treatment did not affect the distribution of either microfilaments or mitochondria (Fig. 5). After 24 h of treatment with BL-I, microfilament configuration was similar to that of controls, in which microfilaments were mainly distributed at the cortex with some microfilaments present in the deeper ooplasm area. Following an additional 20 h of maturation, the treated oocytes matured to MII and the microfilaments were condensed at the oocyte cortex (Fig. 5J), similar to untreated in vitro matured oocytes (Fig. 5H). The mitochondria of both control (Fig. 5C) and BL-I-treated oocytes (Fig. 5E) migrated to the interior of the ooplasm in small groups after 24 h of culture. Similar localization and translocation of both microfilaments and mitochondria in control and BL-I-treated oocytes indicates that delaying nuclear maturation did not disrupt dynamics of cytoplasmic structures and suggests that nuclear and cytoplasmic maturation can be uncoupled without harmful effects on oocyte maturation.
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Results of the MAP kinase activity assay (Fig. 6) indicated that BL-I inhibited the activation of MAP kinase after 24 h of culture, whereas oocytes in the control group reached MI and showed strong activity of MAP kinase. However, 8 h after removal of BL-I, MAP kinase activity of treated oocytes reached a level similar to that of 24 h IVM controls. The peak level of activated MAP kinase was achieved 20 h after BL-I treatment and was the same as that observed in controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Possible Role of MPF in Chromatin Condensationand GVI Synchronization
In vivo, GV of oocytes in maturing follicles are synchronized to a specific stage (GVI) after eCG injection, and further nuclear maturation to the next stages occurs after hCG injection [2, 25]. The mechanism and role of GVI synchronization is unknown. Kubelka et al. [7] suggested that chromosomal condensation is not related to MPF activation because inhibition of MPF activation by inhibiting protein synthesis prevented GVBD but did not influence the process of chromosomal condensation, and 98% of oocytes reached the GVIV stage. Studies in bovine oocytes using the specific MPF inhibitor BL-I have further confirmed this suggestion [13]. However, the present experiment revealed that GV stage distribution of oocytes was unchanged during 24 h culture with 12.5 µM BL-I. The process of chromosomal condensation was completely inhibited by inhibition of MPF activation. The discrepancy between the results of the current study and those previously reported may be due to the use of a nonspecific inhibitor [7] or significantly higher concentrations of BL-I (100 µM) [13, 14, 16]. The specificity of BL-I for inhibition of MPF activation in those reports may be compromised because BL-I also inhibits other important kinases at high concentrations (e.g., the IC50 values for MPF, MAP kinase, protein kinase C, and casein kinase I are 0.68, 94, 160, and 59 µM, respectively) [11].
Roscovitine, another purine derivative that can specifically inhibit MPF activation, can also prevent chromosomal condensation in pig oocytes [26]. The results of the present study confirm those observations and indicate that chromosomal condensation of pig oocyte is dependent on MPF activation. Considering the central role of MPF activation on the resumption and completion of meiotic maturation of oocytes [10, 27, 28], we presume that the in vivo mechanism for synchronizing oocytes at the GVI stage may involve inhibition of MPF activation.
Accelerated Nuclear Maturation after Removal of BL-I
The chronological changes of the nuclear morphology suggest that the inhibitory effect of BL-I on meiotic progression is fully reversible. In the present experiments, pig oocyte maturation was accelerated after removal from BL-I inhibition. In contrast, previous experiments on BL-I-treated bovine oocytes failed to produce accelerated maturation after removal of BL-I [13, 14]. Some MPF activation-related cytoplasmic factors, such as pre-MPF and its up- or downstream factors, may not have been repressed and may have accumulated progressively during BL-I treatment, even though nuclear maturation was suppressed. The presesnt experiments on mitochondria, microfilaments, and MAP kinase activation also indicated uncoupling of cytoplasmic and nuclear maturation.
Normal Cytoplasmic Maturation after Treatmentof Oocytes with BL-I
Mitochondria GVBD is the most dramatic event in resumption of meiosis in oocytes and involves complex nuclear and cytoplasmic changes that impose a great need for energy. The translocation of mitochondria from the oocyte cortex to a deeper area occurs simultaneously with their major functional shift from processing nutrients from cumulus cells to providing ATP to support cytoplasmic and nuclear maturation [21, 29]. MitoTracker Green labeling and confocal microscopy used to examine the mitochondrial distribution revealed that although nuclear maturation was inhibited by 24 h of culture with BL-I, the treated oocytes showed the same mitochondrial translocation pattern as did control oocytes during this period of culture. After removal of BL-I, GVBD occurred in 8 h compared with >20 h in the control group. These treated oocytes reached the MII phase in another 12 h and showed the same pattern of mitochondrial distribution as did untreated in vitro matured oocytes. These results indicate that mitochondrial translocation is independent from nuclear maturation and does not require active MPF. The shorter maturation time required for BL-I-treated oocytes to reach MII could be due to continuous cytoplasmic maturation during the BL-I treatment period, as indicated by translocation of active mitochondria.
Microfilaments Actin is a major component of the cytoskeleton. Disruption of the formation of polymerized actin (microfilaments) resulted in failure of oocyte maturation and embryo development [22]. After 24 h of culture with BL-I, microfilaments were observed as a relatively thick area around the cell cortex and also throughout the cytoplasm. After removal of BL-I and culture for another 20 h, the microfilaments were concentrated at the peripheral area while the oocytes reached the MII stage. Chromosomes were located within the thick microfilament domain of the cortex. The first polar body was seen next to the MII chromosomes with abundant microfilaments. These distribution patterns of microfilaments were the same as those in the untreated oocytes and were consistent with the observations of normal oocyte maturation [22, 30]. These results suggest that the dynamics of microfilament distribution during maturation were not affected by the inhibition of MPF activation with BL-I and might also indicate that normal cytoplasmic maturation continued during and after BL-I treatment.
MAP kinase activation The active forms of MAP kinase, phosphorylated extracellular regulated kinases (pERKs) 1 and 2, were essential to maintain the oocyte at MII [16, 24, 31, 32]. These forms were detected at 18–20 h after IVM, around the time of GVBD initiation, and remained phosphorylated at MII in pig oocytes [16, 24, 31, 32]. High levels of pERK1 and pERK2 are another hallmark of cytoplasmic maturation, in addition to the oocyte's ability to decondense penetrated sperm and to develop to blastocyst after IVF/EC. BL-I treatment will inhibit activation of MAP kinase indirectly as a result of inhibition of p34cdc2 kinase activation [13]. Activation of MAP kinase is inhibited directly by BL-I at a 138-fold concentration of p34cdc2 kinase (IC50 = 94 µM vs. 0.68 µM, respectively) [10]. In the present experiment, inhibition of MAP kinase activation was also observed after 24 h of culture with BL-I. However, only 8 h after removal of BL-I, pERK 1 and pERK 2 levels were elevated to levels similar to those of untreated oocytes matured for 24 h. This accelerated activation of MAP kinase was consistent with the increased kinetics of nuclear maturation observed in experiment 2, in which >66% of the oocytes had undergone GVBD by 8 h after removal of BL-I. At 20 h after removal of BL-I, the peak levels of pERK1 and pERK2 were observed in BL-I-treated oocytes and were not different from levels in controls. This result suggested that BL-I treatment effects were fully reversible and were not detrimental to cytoplasmic maturation.
High Developmental Potential of BL-I-Treated Oocytes after IVF
BL-I-treated oocytes were fertilized and cultured in vitro to clarify possible effects of the inhibitor on fertilization and developmental potential. Asynchronized maturation and aging of early matured oocytes has been suggested as a cause of polyspermy in pig oocytes [33]. In the present study, untreated pig oocytes reached maturation in vitro in 28–44 h, whereas the majority of the BL-I-treated oocytes (94%) reached maturation in the last 8 h of culture, at 36–44 h, but this synchronization did not reduce the polyspermy rate after IVF. These results indicate that asynchronized maturation is not a significant cause of polyspermy in pig IVM/IVF and suggest that bypassing oviduct-related changes in the chemical properties of the zona pellucida during IVM/IVF may be the major cause of polyspermy in pig IVF [34].
At 16 and 24 h after removal of BL-I, the percentage of degenerated oocytes was slightly increased (around 5%) and maturation rate was lowered by 5%. However, the overall fertilization, cleavage, and blastocyst rates were not affected by BL-I treatment. The number of cells per blastocyst tended to be higher after treatment (P = 0.11). When calculated based only on oocytes inseminated instead of total oocytes cultured, the blastocyst rate was 53.3% vs. 46.9% for BL-I-treated vs. control groups, respectively (P < 0.05). Based on these data, we speculate that after removal of BL-I and release from MPF inhibition some oocytes with poor quality may have failed to resume meiosis and degenerated, whereas such oocytes in the control group may have reached MII but were less competent to develop to blastocysts. Synchronization of oocytes at GVI in maturing follicles in vivo might be important for selecting ovulatory follicles and might be MPF activation related.
In the present study, we demonstrated that pig oocytes can be held at the GV stage with BL-I at 12.5 µM concentration and still retain high developmental competence. BL-I treatment may be a very useful way to select higher quality oocytes matured in vitro for IVF, nuclear transfer, and transgenic research and can be applied to the study of mechanisms of oocyte maturation in vitro. Further studies are needed to clarify the role of MPF in vivo in GV developmental synchronization and follicle selection.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by the collaborative animal research program between the University of Missouri Department of Animal Sciences and Monsanto Animal Agriculture Group: Development of Biotechnology Tools for Improved Genetic and Reproductive Performance in Swine. It was also supported in part by the Missouri Agricultural Experiment Station. 
2 Correspondence: Billy N. Day, 159 Animal Science Research Center, Department of Animal Sciences, University of Missouri-Columbia, 920 East Campus Drive, Columbia, MO 65211. FAX: 573 884 7827;dayb{at}missouri.edu
Accepted: January 30, 2002.
Received: December 10, 2001.
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