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a Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After parthenogenetic activation or fertilization, MPF is inactivated in MII oocytes [10–15]. Nonetheless, it is difficult to assess the exact dynamics of MPF activity in fertilized oocytes since the time of sperm penetration varies among individual oocytes. In addition, the data obtained from mice and Xenopus [10, 12–15] may not be applicable to other species. The functions of the Mos-MAPK pathway in oocyte maturation differs between the mouse and Xenopus [16]. Fortunately, sperm-induced oocyte activation can be mimicked by a variety of chemicals as well as by electrical stimulation [17], which allows precise control of the timing of activation. It was suggested that parthenogenetically activated oocytes may provide a suitable model for the study of some aspects of early embryo development [18]. Parthenogenetic development also provides a valuable tool for studying genomic imprinting. Understanding critical molecular components as well as the morphological changes during the initial stage of oocyte activation would be of significance for effective use of activated cytoplasts for cloning by nuclear transfer because successful cloning requires full activation of the recipient cytoplast, but the nuclear transfer process itself does not induce adequate activation of oocytes.
To date, different methods of parthenogenetic activation have been used in an attempt to mimic sperm-induced activation directly or indirectly. Sperm penetration triggers calcium oscillations [19]. The initial calcium increase seems to be important for the initiation of meiotic release. Calcium ionophore A23187 or ionomycin alone was found to induce meiotic release but not pronuclear formation [18,20]; however, A23187 seemed more effective than ionomycin to induce parthenogenetic activation of oocytes ([21], our unpublished results). When used after a calcium-elevation treatment, 6-dimethylaminopurine (6-DMAP) was shown to enhance the activation of young mouse and bovine oocytes [20, 22–24]. Parthenogenetic development similar to in vitro fertilization (IVF) embryo development was obtained by exposure to A23187 sequentially combined with 6-DMAP [17]. The present experiments were designed to identify the changes in MPF and MAPK activities as well as nuclear and microtubule behavior in young and aged bovine oocytes after activation treatments with either A23187 alone or A23187 followed by 6-DMAP. In our experimental design, control oocytes were always cultured parallel with treated oocytes to make sure that changes observed during any particular period were caused by treatments rather than by culture conditions. We report that differential inactivation of MPF and MAPKs occurred in both young and aged bovine oocytes after activation treatments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals used in this study were purchased from Sigma Chemical Company (St. Louis, MO) unless stated otherwise. Gonadotropins used for oocyte maturation were obtained from the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Disease, the National Institute of Child Health and Human Development, and the U.S. Department of Agriculture.
Collection and Culture of Cumulus-Enclosed Oocytes
Bovine ovaries were transported from a slaughterhouse to the laboratory in a thermocontainer at 24–27°C. Collection and culture of oocytes were as described previously [25, 26]. Briefly, oocytes were collected in a 50-ml conical tube by the aspiration of antral follicles (2- to 8-mm diameter) using an 18-gauge needle and a syringe. After being washed three times in Dulbecco's PBS supplemented with 0.1% polyvinyl alcohol, cumulus-enclosed oocytes were selected and washed three times in maturation medium (bicarbonate-buffered M199 with Earle's salts and 25 mM Hepes [Gibco, Grand Island, NY]; and 7.5% fetal calf serum [Gibco] supplemented with 0.5 µg/ml ovine FSH, 5.0 µg/ml ovine LH, and 1.0 µg/ml estradiol). For maturation culture, cumulus-enclosed oocytes were cultured in 100-µl droplets of maturation medium (20–25 oocytes/droplet) covered with mineral oil at 39°C in 5% CO2 and humidified air.
Chemical Activation and In Vitro Culture
At 24-h IVM, oocytes were freed of cumulus cells and placed randomly into two groups. One group of oocytes was activated without further culture (young), and the other was cultured in potassium simplex optimized medium (KSOM) for an additional 16 h before activation treatment (aged). Thus young and aged oocytes refer to oocytes collected, respectively, at 24 h and 40 h of in vitro maturation (IVM) culture. In both the young and aged groups, oocytes with a polar body were selected, and these were activated by 5-min exposure to 5 µM calcium ionophore (A23187) either alone or followed by incubation with 2.5 mM 6-DMAP for 4 h. Treated oocytes were washed and then cultured in 100-µl drops of modified KSOM [27,28] containing 0.1% BSA and 0.4 mM taurine under mineral oil at 39°C in an atmosphere of 5% CO2 in humidified air. Metaphase II (MII) oocytes were cultured under the same conditions to serve as controls. A23187 stock was prepared in dimethyl sulfoxide, and 6-DMAP in sterile distilled water. The chemicals were diluted to the desired concentration in the KSOM before use. Equal numbers of oocytes were sampled in collection buffer at 0, 0.5, 1, 2, 3, 4, and 15 h after treatments and were stored frozen (-70°C) for kinase assays. Alternatively, oocytes were fixed in microtubule-stabilizing buffer for triple staining to visualize the nucleus, microtubules, and actin microfilaments. All experiments were repeated at least twice.
Histone H1 Kinase and MAPK Assay
Histone H1 kinase activity (i.e., MPF activity) and MAPK activity were measured by using histone H1 and myelin basic protein (MBP), respectively, as substrates [3,8, 14, 29]. Five oocytes from each time point were removed from culture, transferred to 4 µl cold (4°C) collection buffer (6.4 mM EDTA, 10 mM NaFl, and 100 mM Na3VO4 in PBS) in Eppendorf tubes, and stored frozen at -70°C. During the kinase assay, the tubes were placed on ice, and 6 µl of homogeneous buffer (HB) was added to each sample (HB: 45 mM ß-glycerophosphate, 12 mM p-nitrophenylphosphate, 20 mM 3-[N-morpholino]-propanesulfonic acid, pH 7.2, 12 mM MgCl2, 12 mM ethylene glycol-bis (ß-aminoethylether) N,N,N1,N1-tetra-acetic acid, 0.8 mM dithiothreitol, 0.1 mM Na3VO4, 20 µg/ml leupetin, and 40 µg/ml aprotinin). The samples were then incubated at 37°C for 15 min. After incubation, 8 µl of kinase buffer (KB) was added (KB: HB with 2 mg/ml histone H1 and 1 mg/ml MBP, 1.2 µM protein kinase inhibitor, and 250 µCi/ml [
-32P]ATP [Dupont, Boston, MA]). Samples were incubated for an additional 30 min at 37°C, and the reaction was terminated by the addition of 18 µl double-strength SDS sample buffer [30]. The samples were heated at 95°C for 5 min and then separated by 8–15% linear gradient SDS-PAGE [17]. Gels were stained with Coomassie Brilliant Blue R250 dye, destained, dried, exposed to Kodak x-ray film and processed for autoradiography. Kinase activities were quantified by measuring the band density of the autoradiograph with an image densitometer (Bio-Rad, Richmond, CA). The kinase activity of every sample within each time point was measured, and an average value was calculated. The histone H1 and MAPK activities in oocytes after 24 h of maturation were arbitrarily set as 100%, and the other values were expressed relative to this activity. The means of histone H1 and MAPK activities were compared with their respective controls by the General Linear Model procedure, using the Statistical Analysis Systems program.
Immunoblotting Analysis of MAPK
Phosphorylation status of MAPK was determined by Western blot analysis of MAP42/extracellular signal-regulated kinase 2 (ERK2) mobility shift with enhanced chemiluminescence (ECL). Fifty oocytes per group were collected in cold SDS sample buffer at 0, 4, and 15 h after activation treatments began and then immediately frozen for storage. After being denatured by boiling for 5 min, the protein samples were separated on 8–15% gradient SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (PVDF; Immobilon-P; Millipore, Bedford, MA). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T), rinsed in TBS-T, and probed with anti-MAP42/ERK2 monoclonal mouse antibody (Transduction Laboratories, Lexington, KY) in TBS-T with 1% BSA. The blot was washed and subsequently incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody diluted appropriately in TBS-T containing 10% normal goat serum. After being washed, the blot was visualized by ECL according to the manufacturer's protocol (Amersham, Buckinghamshire, England).
Immunofluorescence Microscopy of Tubulin, Actin Filament, and Chromatin
Denuded oocytes were fixed and extracted for 30 min at 37°C in a microtubule-stabilizing buffer [31]. The oocytes were washed extensively and blocked overnight at 4°C in the wash medium (PBS supplemented with 0.02% NaN3, 0.01% Triton X-100, 0.2% nonfat dry milk, 2% goat serum, 2% BSA, and 0.1 M glycine). Afterwards, oocytes were incubated with 
- and ß-tubulin mouse monoclonal antibodies (1:200), washed, and then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (1:200; Cappel, West Chester, PA) at 37°C for 2 h. After being washed, the samples were stained for actin filaments with rhodamine-conjugated phalloidin (1:1000; Molecular Probes), washed again, then stained for DNA with Hoechst 33342 (10 µg/ml) in mounting medium containing PBS and glycerol (1:1), and finally mounted onto slides. The samples were observed under an Olympus (Shibuya-Ku, Tokyo, Japan) Provis epifluorescence microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dynamics of MPF and MAPK Activity versus Nuclear and Spindle Changes of Young Oocytes Following Activation Treatments
Young bovine oocytes (24-h IVM) displayed high levels of histone H1 and MAPK activities (Fig. 1A, lane 1, Figs. 3A and 4A). In the control group, the H1 kinase activity in the young MII oocytes remained high through 4 h but decreased by 15 h of control culture, while MAPK activity remained high throughout the 15-h culture period. In young oocytes treated with A23187 alone, a significant decline (about 60%) in H1 kinase activity was observed by 1 h and reached the lowest level (80–90% reduction) at 2–3 h after treatment. However, this activity increased again (3-fold) at 4 h and was detected at a relatively high level similar to that of controls at 15 h (Figs. 1A and 3A). MAPK activity, on the other hand, remained at high levels with minor fluctuations, similar to that of control oocytes throughout the observation period. In oocytes treated with A23187 followed by 6-DMAP, H1 kinase activity decreased significantly (by 70%) by 0.5 h, maintained a basal level from 1 to 4 h, and still remained at a low level at 15 h. In contrast, MAPK activity remained at a relatively high level for the first 3 h, decreased dramatically between 3 and 4 h, and continued at a low level at 15 h after the combined treatment (Figs. 1A and 4A). The inactivation of H1 kinase in A23187-treated oocytes was slower than that observed in the oocytes treated with A23187 combined with 6-DMAP.
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Typical changes in microtubule organization and chromatin morphology are presented in Figure 5. Figure 5A shows the chromosome arrangement of the MII oocytes matured for 24 h (control). When aged in KSOM for 4 h (28-h maturation control), they displayed condensed chromosomes aligned at the metaphase plate over the spindle, which was strongly stained (Fig. 5B). The spindle was lightly stained in the 24-h IVM oocytes (present results and [17]).
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A23187 treatment alone. A23187 treatment of newly matured oocytes (24-h IVM) induced the extrusion of a second polar body and the entry into a subsequent arrest at metaphase known as MIII [13, 20, 32]. At 0.5-h posttreatment, the metaphase chromosomes became more dispersed over the spindle. By 1 h, anaphase II was observed in 77% (n = 22) of the treated oocytes, with separating chromosomes attached to a slightly elongated spindle (Fig. 5C). At 3–4 h, telophase II was reached, and the extrusion of the second polar body was observed (Fig. 5D); moreover, fine microtubule filaments were observed in the cytoplasm. During the release of the second polar body, a large microtubular network and telophase spindles joined the polar body, and a remnant of spindle microtubules existed in the cytoplasm (Fig. 5E). By 15 h, the treated oocytes manifested a transitional stage of metaphase to anaphase structure (named MIII here) with a reduced number of chromosomes spread over a slightly elongated spindle (Fig. 5F).
A23187 plus 6-DMAP treatment. At 0.5–1 h after the combined treatment, chromosome aggregation and spindle destruction was observed: the spindle had completely disappeared; instead, fine microtubule filaments appeared in the cytoplasm, mostly radiating from the clumped chromosome mass (Fig. 5G). At 3 h, microtubules formed networks in the cytoplasm, chromosomes decondensed, and a small pronuclear structure began to form (Fig. 5H). By 4 h, the microtubular filaments intensified, with massive networks visible in the cytoplasm (Fig. 5I). At the same time, a well-defined pronucleus was clearly visible. A fully developed pronucleus (Fig. 5J) and a microtubular network (Fig. 5K) were seen at 15 h after the combined treatment. Control oocytes aged in culture for the same period displayed an elongated spindle, and some had slightly more dispersed chromosomes (Fig. 5L).
Dynamics of MPF and MAPK Activity versus Nuclear and Spindle Changes of Aged Oocytes Following Activation Treatments
For aged oocytes (40-h IVM), compared to young oocytes, treatments by either A23187 alone or A23187 followed by 6-DMAP resulted in a quicker and more dramatic decrease in H1 kinase activity (Figs. 1B, 3B, and 4B). The low activity of H1 kinase remained throughout 0.5-h to 15-h post-treatment with A23187 alone, while an increase in H1 kinase activity was observed by 15 h after treatment with A23187 plus 6-DMAP. Untreated aged control oocytes maintained approximately 70% of the H1 kinase levels found in young (24-h IVM) oocytes. Activity of MAPK remained high in aged oocytes and seemed to increase during control culture. However, in oocytes treated by A23187 alone, MAPK activity remained high for up to 4 h but was significantly reduced at 15 h (Fig. 3B). When A23187 plus 6-DMAP treatment was employed, MAPK activity was reduced significantly at 3 h, and this reduced activity was maintained at 4 and 15 h (Fig. 4B). Once again, as found for the young oocytes, the inactivation of H1 kinase in the aged oocytes also preceded the inactivation of MAPK. The kinase activities were again correlated with the progression of nucleus and microtubules.
A23187 treatment alone. In the untreated aged oocytes, MII chromosomes with elongated spindles appeared in most of the oocytes (Fig. 5L and 6A). At 0.5-h post-treatment, oocytes reached AII, in which chromosomes seemed to be separated over the spindle (Fig. 6, B and C). By 1 h after treatment, telophase was observed in 89% (n = 28) of oocytes (Fig. 6D), with fine microtubule filaments radiating from the spindle (Fig. 6E). At 3–4 h, polar body extrusion was observed, with microtubular networks forming in the cytoplasm (Fig. 6F). By 15 h after treatment, 88% (n = 25) of oocytes were at the pronuclear stage (Fig. 6G). At this time, intense microtubular networks existed. Aged oocytes treated with A23187 underwent the activation process more quickly than young oocytes.
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A23187 plus 6-DMAP treatment. A23187 combined with 6-DMAP induced changes in chromatin and microtubules similar to those observed in young oocytes. However, the cell cycle progression was faster, with chromosome aggregation and spindle destruction evident by 0.5 h after treatment (Fig. 6, H and I). Pronuclear and microtubule network formation began earlier, at 3 h (Fig. 6, J and K), and 45% (n = 29) of oocytes were at mitotic metaphase or anaphase by 15 h after treatment (Fig. 6L).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Metaphase II arrest in mammalian oocytes is probably caused by at least two factors: MPF and cytostatic factor (CSF). It had been believed that the sustained high MPF activity responsible for the MII meiotic arrest was maintained by the CSF activity [33]. However, Watanabe and colleagues demonstrated that cyclin subunits of MPF are degraded before Mos is degraded, and thus MPF activity is inactivated before CSF activity during activation of Xenopus eggs [34]. More recently, it was reported that Mos, a key component of CSF, may regulate MAPK and that the MII-arresting CSF activity of Mos is largely mediated by MAPKs [35–39]. Our present study in activated bovine oocytes demonstrated that inactivation of MPF occurred before inactivation of MAPK. Moreover, in young oocytes activated by A23187 alone, inactivation of MPF occurred whereas MAPK remained high. This evidence suggests that MAPK may have a close relation to CSF or Mos and that MAPK may not be the kinase that stabilizes MPF.
MAPK behavior and function seemed to differ greatly between meiosis and mitosis. Before the first mitotic cleavage, when mitotic spindle organization and chromosome condensation was observed, a sharp rise in MPF activity was observed whereas MAPK activity stayed low, implying that MAPK may not be active in the first mitosis. In other species, MAPK also appears to be specifically activated during meiosis but not in mitosis [10, 13, 40, 41]. However, activation of MPF during the G2/M transition at the first mitosis observed in the present study suggests that MPF is important for the progression of mitosis [6]. The MAPK inactivation was necessary for pronuclear envelope assembly after fertilization in mice [14, 15]. In the present study, during disassembly of the pronucleus and progression to the first mitosis, low MAPK activity was observed in the activated aged oocytes whereas MPF was high, as expected. It was reported that disassembly of the nuclear lamina resulted from phosphorylation of lamin proteins by MPF or cdc2 kinase and that the nuclear lamina appeared to be dephosphorylated upon reassembly around nuclei [42–44]. Present experiments also suggested that an increase in MPF activity, but not MAPK activity, before cleavage in activated aged oocytes is responsible for the disassembly of the pronucleus and mitotic cell cycle progression. However, more study is needed to elucidate the role of MAPK in the first mitotic cell cycle that follows normal fertilization or activation of young bovine oocytes. Inactivation of MPF, induced by A23187 alone, coincided with anaphase and telophase progression, second polar body extrusion, cytoplasmic microtubule filament formation, and finally MIII arrest with an elongated metaphase spindle. A dramatic drop in histone H1 kinase activity was observed around the anaphase-telophase II stage and the time of second polar body extrusion. Histone H1 kinase activity then increased again at 4 h after A23187 treatment and remained at a relatively high level at 15 h. This histone H1 kinase activity rebound is probably responsible for the oocyte's entrance to MIII stage, as previously reported [13, 32, 45]. During this progression, MAPK activity stayed at a high level, suggesting that a spindle microtubular organization might be directly or indirectly regulated by high MAPK activity during the MII-to-MIII transition, as reported previously for mouse oocytes activated by ethanol [13]. During oocyte maturation, MAPK activity also remained high at the MI-to-MII transition in both bovine and mouse oocytes [1, 3]. The incomplete activation of oocytes, resulting in an MII-to-MIII transition (MIII arrest), is very similar to the MI-to-MII transition in both morphological and biochemical aspects [13, 45].
During oocyte aging, H1 kinase activity decreased, yet the chromosomes were maintained at the metaphase II stage with a slightly elongated spindle. This again supports the hypothesis that MAPK but not MPF is responsible for spindle assembly [41, 46]. Furthermore, the fact that aged oocytes but not young oocytes could be fully activated by A23187, leading to pronuclear development, was attributed, at least in part, to the lower H1 kinase activity in aged oocytes and the decrease of MAPK activity after the treatment.
In both young and aged bovine oocytes, meiotic release or resumption can be induced by A23187 treatment, as shown in this study and previously [18]. Moreover, the resumption of meiosis proceeded faster in aged oocytes, in which MPF is already lower than it is in young oocytes. The MPF activity also drops more quickly in aged oocytes than it does in young oocytes when treated by A23187 alone. A majority of the aged oocytes displayed pronuclear formation at 15 h, at which time MAPK dropped dramatically. Again, inactivation of MAPK temporally coincided with chromosome decondensation and pronuclear formation. The release of the second polar body took place in A23187-treated oocytes, whereas the polar body extrusion was inhibited in A23187 plus 6-DMAP-treated oocytes because of the destruction of the spindle; furthermore, chromatin behavior was dissociated from MPF activity, as chromosomes stayed condensed despite an evident drop in H1 kinase activity. However, destruction of the meiotic spindle induced by 6-DMAP occurred before inactivation of MAPK. Also, pronuclear formation is accelerated by 6-DMAP, possibly directly through inhibition of phosphorylation of lamin proteins or indirectly through inactivation of MAPK.
It is known that calcium ionophore treatment can cause a single intracellular calcium rise in MII oocytes [47] and that a likely consequence is the activation of several calcium-dependent proteolytic pathways, leading to the destruction of cyclin B, reduction of MPF activity, and resumption of meiosis. However, in young, newly matured oocytes, because of active synthesis of proteins including cyclin B, a recovery of MPF activity would occur after a single calcium stimulation because of a quick renewal of cyclin B, a re-formation of the MPF complex, and thus a new M-phase arrest, known as metaphase III [32, 45, 48]. A single calcium stimulation can thus cause the temporary inactivation of MPF, but subsequent calcium stimulations are required to destroy the newly synthesized cyclins and related proteins to maintain the inactivation status of MPF [11, 49]. Alternatively, a calcium stimulation followed by treatment with a protein synthesis inhibitor could also cause full activation of the newly matured oocytes [17, 18]. The fact that the calcium elevation destroys cyclin B (and related proteins) and the protein synthesis inhibitor prevents the renewal of those proteins could explain the effectiveness of this combined treatment [17, 18, 26]. Similarly, because most cell cycle regulators such as MPF and MAPKs are phosphorylation-dependent kinases, replacing a protein synthesis inhibitor with a phosphorylation inhibitor such as 6-DMAP in the combined treatment with a calcium stimulator such as A23187 should also be equally effective in inducing activation of oocytes, as demonstrated in this and previous studies [17, 20].
The A23187 plus 6-DMAP treatment led to the inhibition of second polar body extrusion in both young and aged oocytes. Thus, this phenomenon appears to be age-independent and is probably related to the action of 6-DMAP-dependent kinases. In the mouse, 6-DMAP has been shown to both impair the contact between the spindle-pole and the cortex and to inhibit contractile activity in the cortex after activation [22]. In our experiment, instant destruction of the spindle was observed in 6-DMAP-treated oocytes. These processes could lead to the inhibition of polar body extrusion, and therefore, the oocytes would go directly into interphase and only one diploid pronucleus would be formed. The developmental competence of these activated oocytes seems not to be compromised, and a high percentage of oocytes activated in this manner have been shown to develop to the blastocyst stage [17, 20].
In summary, independent inactivation of MPF and MAPKs occurred in bovine oocytes after activation treatments; the inactivation of MPF preceded the inactivation of MAPK. Inactivation of MAPK is associated with the formation of microtubular networks and pronuclear development, while inactivation of MPF coincides with meiotic release. High MAPK activity, instead of MPF activity, seems to be important for the integrity of spindle structures in both young and aged oocytes. Additionally, it is postulated that the activation of MPF, but not MAPK, initiates the first mitosis of activated oocytes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: (860) 486–4375; xyang{at}ansc1.cag.uconn.edu
Accepted: April 14, 1998.
Received: January 21, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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