| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reproductive Technology |
a Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536
b Program in Cell, Molecular and Developmental Biology, Department of Anatomy & Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
developmental biology, gamete biology, meiosis, oocyte development, reproductive technology
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Traditionally, mammalian oocyte cytoplasts are prepared by physically removing nuclear chromatin by micromanipulation techniques in preparation to receive the donor genome [2, 3]. Enucleated oocytes arrested at metaphase of meiosis II (MII) are subsequently "reconstructed" by the addition of the donor karyoplast, typically using either electrofusion [2] or microinjection techniques [4]. Physical enucleation is technically demanding, time-consuming, inherently invasive, and clearly damaging to cytoplast spatial organization. Moreover, development of reconstructed embryos is inherently inefficient. An alternative strategy to physical enucleation has been to treat oocytes with agents that modify the processes of karyokinesis and cytokinesis and result in chemically enucleated oocytes at high rates (>85% [11, 12]). However, Elsheikh and colleagues [13, 14] have reported that exposure of metaphase I and MII oocytes to etoposide, a topoisomerase II inhibitor, and cycloheximide yields enucleated cytoplasts with limited ability to support cleavage or blastocyst development, and to our knowledge, term development of reconstructed embryos has not been reported.
Recently, Baguisi and Overström [15] reported the production of cloned mice from cumulus cell nuclei using cytoplasts enucleated following activation in the presence of a microtubule (MT)-destabilizing drug, demecolcine. Furthermore, this work suggested that demecolcine-induced enucleation may provide cytoplasts of enhanced developmental competence when compared to MII oocytes enucleated by physical means. The reason for enhanced developmental competence remains unclear, but it may result from subtle disturbances in the coordination of cytokinesis and karyokinesis afforded by triggering cell-cycle resumption before exposure to a spindle-disrupting agent. More recently, this demecolcine-induced enucleation protocol has also been applied to the production of cloned mice from embryonic stem cell nuclei [16] and to the production of cloned rabbit fetuses and pigs from adult somatic cells [17, 18].
In the present study, we investigated the temporal consequences of demecolcine-induced enucleation with reference to the cytoskeletal remodeling that occurs during early phases of oocyte activation in CF1 and B6D2F1 mouse strains. In particular, manifestations of demecolcine treatment on spindle rotation/anchoring dynamics and on second polar body (PB) formation and extrusion were investigated.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Hybrid B6D2F1 (C57BL/6 x DBA/2) and outbred CF-1 female mice (age, 8–12 wk; Charles River Laboratories, Wilmington, MA) were used as oocyte donors. Animal care and procedures were conducted according to protocols approved by the Tufts University Institutional Animal Care and Use Committee. A total of 78 B6D2F1 and 66 CF-1 donor females were used. Females were induced to superovulate by i.p. injection of 5 IU of eCG (Calbiochem, San Diego, CA) followed 48 h later by 5 IU of hCG (Calbiochem). The MII oocytes were collected from oviducts 16–17 h after hCG administration in Hepes-buffered KSOM (H-KSOM; no. MR-024-D; Specialty Media, Phillipsburg, NJ) and randomly pooled. Cumulus cells were dispersed by incubation in 150 U/ml of bovine testicular hyaluronidase (Sigma Chemical Co., St. Louis, MO) in H-KSOM at 37°C for 5 min. Cumulus-free oocytes were then washed three times in fresh H-KSOM and immediately activated.
Oocyte Activation, Treatment, and Culture
Oocytes were parthenogenetically activated by a 5-min exposure to freshly prepared 7% (v/v) ethanol in H-KSOM at 37°C and then washed twice in H-KSOM. Removal of oocytes from ethanol was considered as time 0 postactivation (p.a.). To monitor meiotic progression, activated control oocytes (B6D2F1, n = 214; CF-1, n = 256) were cultured for up to 2 h and 15 min and fixed at 30-min intervals starting at 45 min p.a. (ethanol-activated groups). Culture of activated oocytes was at 37°C under 5% CO2 in air in KSOM medium (no. MR-106-D; Specialty Media) containing 1 mg/ml of BSA and amino acids [19].
Other activated oocytes were treated with the MT-destabilizing drug demecolcine (Sigma) at a concentration of 0.4 µg/ml in KSOM and comprised the Deme treatment groups. These oocytes were cultured in the continued presence of demecolcine starting either immediately after activation (Deme 0 groups; B6D2F1, n = 200; CF-1, n = 199) or with a delay of 5 min (Deme 5 groups; B6D2F1, n = 210; CF-1, n = 202), 10 min (Deme 10 groups; B6D2F1, n = 208; CF-1, n = 207), or 15 min (Deme 15 groups; B6D2F1, n = 198; CF-1, n = 205) after their removal from ethanol. Demecolcine-treated oocytes were fixed at 30-min intervals, from 45 to 135 min p.a., identical to control oocytes. To determine oocyte meiotic status at the onset of demecolcine treatments, some control activated B6D2F1 (n = 251) and CF-1 (n = 267) oocytes were fixed at the same times that drug exposure was initiated in the treatment groups: 0, 5, 10, and 15 min p.a.
Activation and demecolcine treatments were repeated at least three times on separate days. Approximately 50 oocytes were examined per treatment at each defined time-point.
Fixation of Oocytes and Processing for Immunofluorescence Analysis
At defined time-points after activation, control and demecolcine-treated oocytes were fixed and extracted for 30 min at 37°C in a MT-stabilizing buffer containing 0.1 M PIPES, 5 mM MgCl2, 2.5 mM EGTA, 3.7% (v/v) formaldehyde, 0.1% (v/v) Triton X-100, 1 µM taxol, 0.01% aprotinin (w/v), 1 mM dithiothreitol, and 50% (v/v) deuterium oxide. Fixed oocytes were stored until processing at 4°C in a PBS blocking solution containing 1% (w/v) BSA, 0.2% (w/v) powdered milk, 2% (v/v) normal goat serum, 0.1 M glycine, 0.2% (w/v) sodium azide, and 0.01% Triton X-100 [20].
A triple-labeling protocol was used for the detection of MTs, microfilaments, and chromatin by fluorescence microscopy [21]. Oocytes were first incubated for 1 h at 37°C in a mixture of mouse monoclonal anti-
-tubulin and anti-ß-tubulin antibodies (Sigma) at a 1:1000 final dilution. After several washes in 0.1% polyvinylpyrrolidone (PVP)/PBS at room temperature, oocytes were incubated at 37°C in PBS blocking solution for 30 min and then in a 1:150 dilution of a donkey anti-mouse fluorescein-conjugated immunoglobulin G (Jackson ImmunoResearch, West Grove, PA) for 45 min at 37°C. Oocytes were washed again several times in 0.1% PVP/PBS and incubated at 37°C for 30 min in 10 U/ml of Texas Red-conjugated phalloidin (Molecular Probes, Eugene, OR) to stain actin filaments. Finally, after extensive washing in 0.1% PVP/PBS, oocytes were incubated at room temperature for 15 min in 10 µg/ml of Hoechst 33258 (Molecular Probes) and mounted in 50% glycerol/PBS containing 25 mg/ml of sodium azide.
Labeled oocytes were examined using a Zeiss IM-35 inverted epifluorescence microscope (Zeiss, Thornwood, NY) fitted with filters selective for Hoechst, fluorescein, and Texas Red and a 50-W mercury lamp. Selected images were acquired using a Photometrics Cool Snap CCD camera (Roper Scientific, Inc., Trenton, NJ) running on Metamorph software (version 5.0; Universal Imaging Corp., Downington, PA).
Statistical Analysis
Data were analyzed by chi-square test or Fisher exact test. A probability value of P < 0.05 was considered to be statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ethanol-Activation Rates Are Not Affected by Demecolcine Treatment
The effect of demecolcine on the meiotic spindle was evident 15 min after the onset of treatment, because spindles in treated oocytes were smaller and displayed a lower MT density compared to untreated control activated oocytes. Although MT density decreased with extended exposure to the drug, spindle MTs did not disappear completely, and even after 2 h of treatment, a few short MTs were detected in the majority of oocytes.
At 45 min p.a., 98% and 92.5% of the control activated B6D2F1 and CF-1 oocytes, respectively, had resumed meiosis, as evidenced by chromatid segregation, spindle elongation, and the presence of a large, actin-rich cortical protrusion or, in a few cases, a completely extruded second PB. These oocytes were considered to be activated (Fig. 1A). Similar rates of activation at 45 min p.a. (80–98%) (Table 1) were observed in all groups of demecolcine-treated oocytes according to the same criteria, except that spindle elongation failed to occur. Although the extent of chromosome separation was reduced due to spindle disruption, two distinct clusters of chromosomes were clearly visible in these treated activated oocytes, indicating that an effective anaphase had occurred. The chromosomes were subcortical to the oocyte cortex and usually connected by a spindle remnant that resembled a midbody (Fig. 1B). As in the control group, a small fraction of oocytes had already extruded a second PB. Interestingly, a single group of chromosomes and no detectable MTs were present in treated oocytes that failed to activate (Fig. 1C).
|
|
In the B6D2F1 strain, activation rates of demecolcine-treated oocytes were equivalent to those of nontreated control oocytes at all time-points examined (Table 1). Rates of activation in some groups of CF-1 treated oocytes were lower than in the control group at 75 and 105 min p.a. However, this effect was transitory and reversible, because at 135 min after ethanol exposure, activation rates were again equivalent among all groups. Therefore, normal rates of activation are obtained when activated oocytes are cultured in the continuous presence of demecolcine.
Effect of Demecolcine on Spindle Rotation in Activated Oocytes
As noted by others [22], two cortical protrusions formed adjacent to each spindle pole shortly after activation in nontreated control oocytes. One protrusion then regressed as the spindle rotated toward the remaining protrusion and assumed an orientation perpendicular to the plasma membrane (Fig. 2). Eventually, this structure was constricted at the oolemma and gave rise to the second PB. Activated oocytes treated with demecolcine yielded two classes that displayed either a single (type A oocyte) or a double (type B oocyte) cortical protrusion overlying the remnants of the spindle (Fig. 3). The two sets of chromosomes were closer to each other in type A oocytes than in type B oocytes, suggesting that the formation of one or two protrusions was probably dependent on the extent of meiotic cell-cycle progression before spindle disruption. Consistent with this idea, type A oocytes were more frequently observed when demecolcine treatment started immediately or 5 min after activation (Deme 0 and Deme 5 groups, respectively), whereas the incidence of type B oocytes predominated when treatment was delayed for 10 or 15 min (Deme 10 and Deme 15 groups, respectively; data not shown).
|
|
Initiation of spindle rotation occurred in all groups of demecolcine-treated oocytes, except for the Deme 0 group in the CF-1 strain, but at lower rates than in control activated oocytes (Table 2). Although only a few short spindle MTs were present in treated oocytes, orientation of spindle remnants and the two chromosomal sets relative to the plasma membrane was used as an indicator of spindle rotation. The CF-1 oocytes treated with demecolcine consistently exhibited a comparatively low percentage of activated oocytes undergoing a partial or complete spindle rotation at all p.a. time-points examined. Demecolcine also impaired spindle rotation in B6D2F1 oocytes at 45 and 75 min p.a. compared to the controls, but the effect was less pronounced than in CF-1 oocytes. However, a dramatic decrease in the percentage of treated B6D2F1 oocytes showing spindle rotation occurred in all treatment groups at 105 and 135 min p.a., suggesting that spindle rotation was reversed with prolonged drug exposure. By 135 min p.a. oocytes showing complete spindle rotation were observed in only 0–9.6% of the CF-1 and B6D2F1 treated oocytes, compared to 100% in both control groups, and the lack of differences between demecolcine treatments further attested to the effectiveness of demecolcine on spindle rotation. In all control and treated oocytes showing a completely rotated spindle at 135 min p.a., extrusion of the second PB had occurred. Together, these results indicate that continued exposure to demecolcine after oocyte activation inhibits spindle rotation independent of the time of initiation of the treatment and the strain of the oocyte, although the kinetics of this inhibition varies between strains.
|
Complete Extrusion of the Second PB Is Inhibited in the Presence of Demecolcine
Whereas the onset of second PB formation was evident in all treated activated oocytes, forming one or two cortical protrusions overlying chromosomes, completion of second PB extrusion was impaired in the presence of demecolcine. By 45 min p.a., a small and similar percentage of activated control and treated oocytes displayed a completely extruded second PB (Fig. 4). The rates of PB extrusion in B6D2F1 and CF-1 control oocytes increased progressively with time, reaching 100% at 135 min p.a., but complete PB extrusion in demecolcine-treated oocytes from both strains was significantly decreased, with rates ranging from 23.1% to 70.2% at the various p.a. time-points examined. Even though some differences were detected between treatments in both strains, a correlation between rates of second PB extrusion and the onset of demecolcine treatment could not be established. On the other hand, comparison of second PB extrusion rates in demecolcine-treated oocytes from both strains revealed significant differences between Deme 10 groups at 75 min p.a., between all treatment groups at 105 min p.a., and between Deme 0, Deme 10, and Deme 15 groups at 135 min p.a. These results suggest a strain-dependent effect of demecolcine on the suppression of second PB extrusion, being more pronounced in oocytes from the CF-1 strain.
|
In those demecolcine-treated oocytes that failed to extrude a second PB, cortical protrusions enlarged over time and, in some oocytes, showed signs of constriction at the oolemma (Fig. 5). To determine whether PB extrusion was merely delayed in these oocytes, they were cultured for a longer period of time (4 h) before fixation and analysis. In most of the oocytes, the cortical protrusions were reabsorbed, whereas the formation of two pronuclei indicated that the cell cycle progressed to early interphase.
|
Characteristic Phenotypes Are Observed in Activated Oocytes Treated with Demecolcine that Complete Second PB Extrusion
All control activated oocytes extruded a second PB containing half of the chromosomal complement and displayed a midbody perpendicular to the plasma membrane. This phenotype was classified as type C (Fig. 6A) and was also observed in a fraction of demecolcine-treated oocytes with completely extruded second PBs. However, midbodies in treated type C oocytes were narrower and shorter than in control activated type C oocytes and were defined as midbody-like structures. Other treated oocytes that completed second PB extrusion displayed characteristic phenotypes that were never detected in control activated oocytes. Type D oocytes (Fig. 6B) deployed one set of chromosomes in the oocyte cytoplasm and one set inside the extruded second PB, connected by a midbody-like structure as in type C oocytes. However, in type D oocytes, the midbody-like structure was oriented in parallel to the plasma membrane, indicating that spindle rotation had not occurred. Moreover, a prominent protuberance adjacent to the second PB was present in type D oocytes, probably caused by the subcortical position of the chromosomal complement in the oocyte in the absence of spindle rotation. Other demecolcine-treated oocytes displayed two (type E) (Fig. 6C) or one (type F) (Fig. 6D) completely extruded second PBs that contained all chromosomes. Therefore, types E and F represent totally enucleated oocytes. Rotation of the spindle had not occurred in these oocytes either, as evidenced by the parallel orientation of the remaining spindle MTs and the two sets of chromosomes inside the PB relative to the plasma membrane.
|
The frequency of each of these phenotypes varied according to the onset of the demecolcine treatment with regards to activation, duration of treatment, and strain of oocyte, further indicating variability in the responsiveness to demecolcine (Fig. 7). In B6D2F1 oocytes, type C was the most frequent at 45 and 75 min p.a. in all treatments, except for the Deme 0 group, in which it was detected at a rate similar to that of type D. Prolonged exposure, independent of when treatment was initiated, caused a shift to the type D phenotype, as seen by the high percentage of type D oocytes at 135 min p.a. in all treatments. The CF-1 oocytes exhibited a strikingly different response. For all treatments, type F was the main phenotype in those oocytes that completed second PB extrusion by 45 min p.a. An increase in the frequency of type D oocytes was observed over time, and at 135 min p.a., type D and type F oocytes appeared at a similar frequency, except for the Deme 5 group, in which most of the oocytes were still of type F.
|
Treatment of Activated Oocytes with Demecolcine Induces Enucleation in a Strain-Dependent Manner
According to the previous results, most CF-1 oocytes that completed extrusion of the second PB were enucleated as a result of demecolcine exposure, whereas the majority of treated B6D2F1 oocytes retained half of the chromosomal complement. This result is summarized in Figure 8, which shows the combined results from all time-points examined for the total oocytes enucleated as a result of the various demecolcine treatments applied. Enucleation rates ranging from 48.3% to 76.7% were obtained in those CF-1 oocytes that completed second PB extrusion and from 1% to 17.3% in the B6D2F1 strain (Fig. 8A). In the four treatments applied, the rates of enucleation were significantly higher in CF-1 than in B6D2F1 oocytes, indicating that the efficiency of demecolcine in inducing oocyte enucleation was strain-dependent. Moreover, enucleation efficiency was also dependent on the time at which treatment was initiated p.a., as indicated by the higher enucleation rates obtained in both strains of oocytes when demecolcine treatment was initiated sooner rather than later after activation.
|
Because of the low rates of complete PB extrusion in demecolcine-treated oocytes, a dramatic decrease in the rates of enucleation is observed when the total activated oocytes are considered (Fig. 8B). Maximum enucleation rates of only 21% and 6.9% in CF-1 and B6D2F1 oocytes, respectively, were obtained, and again, the enucleation efficiency in all four treatments was higher in CF-1 than in B6D2F1 oocytes. Although enucleation rates of the total activated oocytes were equivalent between treatments in CF-1 oocytes, some differences were detected between B6D2F1 oocytes subjected to different treatments. These differences indicated, again, that exposure to demecolcine early after activation results in higher rates of enucleation than when the treatment is delayed.
Meiotic Cell-Cycle Progression after Activation Is Strain-Dependent
To determine if the different efficiencies of demecolcine for inducing enucleation in CF-1 and B6D2F1 oocytes could be related to variations in the oocyte meiotic progression after activation, some control activated oocytes were fixed at the same time-points p.a. at which the demecolcine treatments were initiated. Because the effects of demecolcine on the meiotic spindle are not immediate, the meiotic progression at 45 min p.a. was also recorded. Although the time course of activation was similar in oocytes from the two strains, the rate of cell-cycle progression after the activation stimulus was slightly different (Table 3). Release from MII arrest and entry into anaphase followed a similar progression in the two groups of oocytes after ethanol exposure, but the anaphase-telophase transition proceeded faster in CF-1 oocytes. Thus, 2.6% and 4.4% of activated CF-1 oocytes were at telophase II at 10 and 15 min p.a., respectively, whereas all activated B6D2F1 oocytes remained at anaphase II. By 45 min p.a., 87.1% of activated CF-1 oocytes had entered telophase II, a value significantly higher than the 60% observed for activated B6D2F1 oocytes.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Resumption of meiosis after fertilization or artificial activation of MII-arrested oocytes is characterized by chromosome segregation to the spindle poles, elongation and rotation of the meiotic spindle, and extrusion of a second PB containing half the chromosomal complement of the oocyte. M-phase exit is triggered by the inactivation of maturation-promoting factor (MPF), and it is now well established that cyclin B degradation (and, thus, MPF inactivation) requires an intact spindle [23]. Consistent with this, MII oocytes treated with demecolcine or nocodazole before in vitro fertilization or parthenogenetic activation remain arrested in the M phase despite the occurrence of a normal pattern of calcium oscillations [24, 25]. The exact mechanism by which the meiotic spindle mediates the transition from meiotic M phase to embryonic interphase remains unclear. In the present study, oocytes activated with ethanol before demecolcine treatment exhibited activation rates comparable to those of activated control oocytes never exposed to demecolcine. Ethanol exposure induces an immediate increase in intracellular calcium [26] and rapid progression into anaphase, as evidenced by the rapidity of meiotic cell-cycle resumption in control activated oocytes from the two strains analyzed in this work. Because a delay exists between the onset of demecolcine application and detectable signs of spindle MT disruption, it is not surprising that the acute effects of ethanol on cell-cycle resumption are not impeded. In fact, as our results in control activated oocytes show, most oocytes (
60%) exited M phase and progressed to anaphase by the end of the 5-min ethanol exposure (0 min p.a.). Therefore, most of the oocytes were already at anaphase II or the anaphase-telophase transition when the demecolcine treatment was applied. We show here that when demecolcine is applied after the activation stimulus, activation proceeds in the presence of the drug. However, at later stages, clear consequences of demecolcine exposure occurred that altered the relationship between karyokinesis and cytokinesis.
Demecolcine binds tightly to tubulin dimers and prevents MT polymerization, resulting in the loss of dynamic spindle MTs in mitotic and meiotic cells. Immunofluorescence staining with antitubulin antibodies allowed us to confirm the time course and extent of spindle disruption by demecolcine and, furthermore, showed that few short MTs remain in the majority of oocytes even after prolonged (2-h) drug exposure. The presence of these spindle remnants may reflect differential stability of some MTs in the spindle, and they likely correspond to interpolar MTs [27]. Not surprisingly, spindle disruption impaired the extent of chromatid segregation under these conditions. However, because oocytes were activated before demecolcine treatment, the variable degrees of chromosome segregation that we observed most likely resulted from the time of demecolcine administration, its uptake kinetics, and variations in anaphase onset or duration. These results establish that cell-cycle activation occurs before gross disruptions of spindle stability.
In early telophase, the meiotic spindle rotates from a parallel to a perpendicular orientation relative to the plasma membrane coincident with the initiation of second PB formation (Fig. 2) [22]. Although the mechanism of spindle rotation is unclear, the presence of an actin-rich cortical domain overlying the spindle [28], coupled with the inhibition of spindle rotation in both mouse and Xenopus oocytes treated with cytochalasin [28, 29], suggests that the interaction of spindle MTs with actin filaments of the cell cortex mediates spindle rotation and coordinates karyokinesis and cytokinesis. Consistent with this, disruption of the spindle should also inhibit its rotation, as our results with demecolcine demonstrate. In fact, some demecolcine-treated oocytes undergo some degree of spindle rotation, especially in the case of B6D2F1 eggs, but the process is completed in less than 10% of the oocytes. These observations suggest that spindle rotation is initiated before demecolcine induces depolymerization of the spindle MTs, which as a result perturbs interactions between MTs and cortical microfilaments and impairs further rotation of the spindle. Although suppression of spindle rotation occurred in both strains of oocytes examined, the percentage of oocytes with partially or completely rotated spindles at 45 and 75 min p.a. was higher in the B6D2F1 than in the CF-1 strain. This result indicates strain-dependent variation in the kinetics of inhibition of spindle rotation induced by demecolcine that cannot be related to interstrain differences in the initiation and progression of spindle rotation or the rate of cell-cycle progression (as detected in control activated oocytes from the two strains). Thus, these strain-dependent variations may result from other factors associated with elongation and anchoring of the spindle, such as centrosome positioning [30].
As our results show, an additional effect of demecolcine on activated oocytes was inhibition of second PB extrusion. The initial phase of PB formation, described by Maro et al. [28] within in vitro-fertilized mouse eggs as a "furrowing" of the plasma membrane in the region overlying the spindle, was observed in most demecolcine-treated oocytes. This was evidenced by the formation of one or two actin-rich cortical protrusions. However, later "furrow constriction" and abscission was generally impaired in oocytes activated in the presence of demecolcine. In fact, nocodazole or demecolcine treatment before furrowing and cleavage in sea urchin eggs has shown that MTs are required for furrow stimulation and formation of the actomyosin contractile ring; however, once furrowing has been stimulated, MTs are unnecessary [31]. This and other studies in mammalian tissue culture cells have also demonstrated that MTs are essential for abscission, because depolymerization of the central spindle in late anaphase blocks the completion of cytokinesis [32]. Several proteins necessary for cytokinesis have been localized to the central spindle [33], and it has been suggested that MTs may serve as tracks along which these proteins and other components of the cell move into the cleavage furrow [34–36]. Specifically, the presence of a functional midbody is required in mammalian cells to complete division. Formation of the midbody begins in anaphase, when MT bundles assemble in the central spindle, but functional midbody assembly also requires formation of new MTs nucleated by 
-tubulin centers during telophase [37, 38]. In view of this, suppression of new MT polymerization would be expected in demecolcine-treated oocytes and could underlie the inhibition of second PB extrusion. Interestingly, midbody-like structures were detected in some of the treated oocytes that completed second PB extrusion, and specifically in all type C and type D oocytes. Because MTs that form the central spindle and the midbody are extremely stable [39], it is possible that some MT bundles could assemble in these oocytes before extensive MT depolymerization, forming a midbody-like structure that persisted. However, detection of these midbody-like structures in oocytes with a completely extruded second PB argues against the need for newly nucleated MTs in the completion of cytokinesis, unless this was not required for PB abscission or a different mechanism was used in these oocytes to complete division. In fact, second PB extrusion in type E and type F oocytes was completed in the absence of a midbody or a midbody-like structure. Spindle rotation had not occurred in these oocytes, and the spindle remnants, together with all chromosomes, were extruded inside the second PB, leaving an enucleated oocyte. Interestingly, chemically enucleated mouse oocytes produced by a combined treatment with etoposide and cycloheximide also extrude PBs containing all oocyte chromosomes without involvement of the spindle [12]. Completion of cytokinesis in the absence of MTs has also been reported in other studies [31, 37], and a midbody-independent mechanism for cytokinesis has been proposed to exist in mammalian cells [40]. Thus, it is also possible that in all or some of the demecolcine-treated oocytes that completed second PB extrusion, this alternative mechanism was used because of the absence of a midbody or the presence of a nonfunctional midbody-like structure. The mechanism of PB extrusion and its dependence on midbody integrity will require further study.
Suppression of second PB extrusion in the presence of demecolcine was independent of the time of treatment but was dependent on the strain of the oocytes tested. In general, the incidence of second PB extrusion was lower in treated CF-1 than in treated B6D2F1 oocytes. Almost all treated B6D2F1 oocytes with an extruded second PB were of type C or type D and exhibited a midbody-like structure. On the other hand, as our results in control activated oocytes show, extrusion of the second PB seems to proceed somewhat faster in the B6D2F1 strain (Fig. 4). Therefore, strain-specific variations in the time course of midbody formation and second PB extrusion may explain the observed differences between treated CF-1 and treated B6D2F1 oocytes.
As expected from previous work [15], timely perturbation in spindle function during second PB extrusion also resulted in oocyte enucleation. Inhibition of spindle rotation and the extent of chromosome migration in the presence of demecolcine probably contributed to the expulsion of the entire chromosome complement inside one or, occasionally, two second PBs. As our results show, the onset of the demecolcine treatment in relation to activation is key to achieving enucleation. Application of demecolcine either immediately or a few minutes after ethanol exposure results in higher enucleation rates than application of the drug 15 min after activation, which suggests that the extent of chromatid segregation is a key determinant of enucleation. In addition, a strain effect was also observed for enucleation efficiency, but the reasons for this are unclear. If the proximity of the two groups of chromosomes was decisive for enucleation, then slower progression into telophase after activation would favor enucleation. However, a faster anaphase-telophase transition was observed in control activated oocytes of the CF-1 strain, with higher rates of enucleation in all demecolcine treatments, than was observed in the B6D2F1 strain, with lower rates of enucleation. Thus, other parameters must account for the strain-dependent efficiency of enucleation. Further studies are needed to better understand the mechanisms underlying demecolcine-induced enucleation before this technique can be efficiently applied for oocyte enucleation in various strains and species.
The majority of CF-1 oocytes treated with demecolcine that completed second PB extrusion were enucleated, and enucleation rates close to 80% were obtained. However, because many activated oocytes failed to complete second PB extrusion, the overall enucleation efficiency approximated 20%. Therefore, at least in the CF-1 strain, impairment of PB extrusion is a limitation to enucleation. Similar results have been obtained in demecolcine-treated goat and bovine activated eggs [41, 42]. Shorter treatments with demecolcine, which would allow MT regeneration by late telophase, may promote the completion of second PB extrusion. In fact, preliminary studies with oocytes exposed to demecolcine for only 15, 30, or 45 min resulted in slightly higher rates of second PB extrusion, but the rates of oocyte enucleation were also reduced (unpublished results). Because the effects of demecolcine on MT depolymerization and regeneration are not immediate with respect to time of application and removal, synchronization of treatment with oocyte cell-cycle stage may be difficult to achieve. Possibly, the use of other MT-disrupting drugs that have more rapid and reversible effects, such as nocodazole, may provide better control over the integration of cytokinesis and karyokinesis.
In conclusion, we have shown that culture of activated mouse oocytes in the presence of demecolcine results in normal rates of oocyte activation and progressive cytoskeletal changes after activation. Disruption of spindle MTs by demecolcine impairs chromosome migration, suppresses spindle rotation, inhibits second PB extrusion, alters chromosome partitioning, and thereby, results in the generation of enucleated oocytes. Enucleation efficiency depends on both the onset of the demecolcine treatment in relation to oocyte activation and the genetic background of the oocyte. Further studies are needed to better characterize the mechanism of demecolcine-induced enucleation to establish a more efficient protocol for the enucleation of oocytes of various strains and species in nuclear transfer procedures. In addition, because MTs play a key role in many cellular processes during embryonic development, the potential side effects of MT-disrupting drugs on development of reconstructed embryos must be examined. However, production of live cloned mice from both cumulus and embryonic stem cell nuclei [15, 16] using demecolcine-enucleated cytoplasts demonstrates the potential of this new technique for nuclear transfer practice and indicates that, when used in this way, long-term effects may be minimized.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence. FAX: 508 839 7091; eric.overstrom{at}tufts.edu
3 Current address: Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
Received: 12 June 2002.
First decision: 1 August 2002.
Accepted: 21 October 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-tubulin. Dev Biol 2001 239:281-294[CrossRef][Medline]-Tubulin participates in the formation of the midbody during cytokinesis in mammalian cells. J Cell Sci 1993 105:145-156[Abstract]-tubulin at the midbody is required for the completion of cytokinesis during the mammalian cell division. J Cell Sci 1995 108:2955-2962[Abstract]This article has been cited by other articles:
|
|
 |
|
  E. Ibanez, D. F Albertini, and E. W Overstrom Effect of genetic background and activating stimulus on the timing of meiotic cell cycle progression in parthenogenetically activated mouse oocytes Reproduction, January 1, 2005; 129(1): 27 - 38. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
