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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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In a previous study, we observed that blastocyst development rates were similar when oocytes were activated chemically or by in vitro fertilization (IVF) [4]. Despite many reports on bovine oocyte activation, the molecular mechanisms involved in the initial activation process stimulated by different sequentially combined treatments remain unclear. The increase in the activities of maturation-promoting factor (MPF), as well as mitogen-activated protein kinase (MAPK), was found to be necessary for the onset of germinal vesicle breakdown (GVBD) and metaphase progression during oocyte maturation and meiotic arrest [9–14]. MPF is composed of cyclin B and p34cdc2 kinase, and it displays a cyclic activity that peaks at metaphase [15]. After parthenogenetic activation or fertilization, MPF is inactivated in metaphase II (MII) oocytes [16–20], and MAPK cascade has been shown to play a crucial role in regulating meiotic cell cycles during oocyte maturation, metaphase arrest, and early embryonic development [11, 21–26]. However, the role of MAPK in the first embryonic cell cycle is not well understood. It has been shown that multiple electrical pulses and combined treatments of chemicals improve the developmental ability of oocytes and that effective activation methods are those that mimic the intracellular changes, particularly calcium oscillations, during fertilization [27–31]. However, the molecular mechanisms underlying such calcium changes are not fully understood in most mammalian species.
The present study consisted of experiments to determine the treatment effects on cell cycle regulation in the activation of bovine oocytes by various agents that increase intracellular calcium as well as inhibitors of protein synthesis or phosphorylation. Oocytes activated by sperm with our standard IVF protocol were also examined simultaneously for direct comparisons with the parthenogenetically activated oocytes. The results demonstrated that different activation protocols resulted in different kinetics in cell cycle proteins and kinase activities during progression of the first cell cycle in the activated bovine oocytes.
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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-Oocyte Complexes
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 [1, 32]. Briefly, cumulus-oocyte complexes (COCs) were collected in a 50-ml conical tube by aspiration of antral follicles (2–7-mm diameter) using an 18-gauge needle and a syringe. After washing three times in Dulbecco's PBS supplemented with 0.1% polyvinylalcohol (DPBS-PVA), COCs were selected (more than two layers of cumulus) and washed three times in maturation medium. For maturation culture, COCs were cultured in 100-µl droplets of maturation medium (20–25 oocytes per droplet) covered with mineral oil at 39°C in 5% CO2 and humidified air. The maturation medium was M199 with 25 mM sodium bicarbonate, Earle's salts, and 25 mM Hepes (Gibco, Grand Island, NY), supplemented with 7.5% fetal calf serum (Gibco), 0.5 µg/ml ovine FSH (oFSH), 5.0 µg/ml ovine LH (oLH), 1.0 µg/ml estradiol, and 0.25 mM pyruvate.
Chemical Activation and In Vitro Culture
After maturation culture for 24 h, oocytes were stripped of their cumulus cells by vortexing. Denuded oocytes with a polar body were selected and then assigned to the following treatments: calcium ionophore A23187 (A23187, 5 µM for 5 min) either alone or followed by incubation in 6-DMAP (2.5 mM for 4 h), or CHX (10 µg/ml for 6 h), or CHX plus cytochalasin D (CD, 2.5 µg/ml for 6 h). The A23187 and CD stocks were prepared in dimethyl sulfoxide; 6-DMAP and CHX stocks in sterile distilled water. All the chemicals were diluted to the desired concentration in KSOM [33, 34] supplemented with 0.1% BSA (Sigma; A-9647) before use. After single or combined treatment, oocytes were washed and then cultured in 100-µl drops of KSOM containing 0.1% BSA under mineral oil at 39°C in an atmosphere of 5% CO2 in humidified air. Metaphase II oocytes without activation treatment were cultured under the same conditions as controls.
IVF of Bovine Oocytes
IVF procedure was described previously [32]. Briefly, frozen semen was thawed in a 37°C water bath. The sperm were washed twice by centrifugation with washing medium, which was based on Brackett's defined medium [32, 35], and then capacitated with heparin and coincubated for 6 h with cumulus-enclosed oocytes in Brackett's fertilization medium [32]. Afterward, the fertilized oocytes were denuded of cumulus cells as in the activation experiment, washed, and then cultured in KSOM medium as described above.
Histone H1 Kinase and MAPK Assay
Histone H1 kinase activity (i.e., MPF activity) and MAPK activity were analyzed as previously described [13, 18, 36]. Briefly, five oocytes from each time point were removed from culture and transferred to 4 µl cold (4°C) collection buffer (DPBS-PVA added with 6.4 mM EDTA, 10 mM NaFl, and 10 mM Na3VO4) in Eppendorf (Hamburg, Germany) tubes and stored frozen at -70°C. Detailed protocols for kinase reaction with substrates, gel electrophoresis, autoradiography, and quantification of histone H1 kinase and MAPK have been described by Liu et al. [18]. The histone H1 and MAPK activities in oocytes after 24 h of maturation were arbitrarily set at 100%, and the other values were expressed relative to this activity.
Western Blot Analysis
The relative amounts or phosphorylation status of cyclin B1, cdc2, cdc25 and MAPK were determined by Western blot analysis with enhanced chemiluminescence. Fifty oocytes per group were collected at 0, 1, 4, and 15 h following activation treatments in cold SDS sample buffer and then immediately frozen for storage. After being denatured by boiling for 5 min, the protein samples were separated on a 8–15% gradient SDS-PAGE and transferred onto polyvinylidene fluoride membrane (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 in a sealed plastic bag at room temperature for 1.5 h or 4°C overnight, with either cyclin B1, cdc2 (Sigma), or cdc25 and anti-MAP42/ERK2 antibody (Transduction Laboratories, Lexington, KY) diluted in TBS-T added with 1% BSA. The antibody dilution for cyclin B1, cdc2, cdc25, or ERK2 was 1:200, 1:500, 1:250, or 1:500, respectively. The blot was washed and subsequently incubated with horseradish peroxidase-conjugated IgG secondary antibody diluted appropriately in TBS-T containing 10% normal goat serum. After being washed, the blot was visualized by enhanced chemiluminescence according to the manufacturer's protocol (Amersham, Buckinghamshire, UK).
Immunofluorescence Microscopy
Sampled oocytes were fixed for 30 min at 37°C in a microtubule-stabilizing buffer [37, 38]. The oocytes were washed extensively and blocked overnight at 4°C in the wash medium, which was DPBS 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. Afterward, oocytes were incubated with anti-
and -ß tubulin mouse monoclonal antibody (1:200), washed, and then incubated with fluorescein isothiocyanate-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, Eugene, OR) and washed again; they were 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 (Tokyo, Japan) Provis epifluorescence microscope.
Microinjection of Anti-ERK2
Oocytes treated with A23187 for 5 min were microinjected 4 h later in Ca2+-free PBS supplemented with 0.1% BSA. Micromanipulations were performed with Narishige (Tokyo, Japan) manipulators mounted on a Nikon (Garden City, NY) inverted microscope. Each oocyte was injected with approximately 10 pl of the anti-ERK2 (1:50) or buffer solution as control. The oocytes were washed, cultured in KSOM medium, and then assessed for activation and pronuclear development at 15 h after A23187 treatment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this experiment, systematic comparisons of oocyte nuclear and cytoskeletal morphology were made following different activation regimes and IVF. Typical morphological changes in artificially activated and IVF oocytes are summarized in Table 1. The typical morphologies, such as anaphase, second polar body extrusion, spindle destruction, and chromosome aggregation were described in detail in our recent paper [18]. The results showed that A23187 treatment alone induced meiotic release and led to anaphase-telophase transition but finally arrested at metaphase III (MIII) stage (73%), with a reduced number of chromosomes spread over a slightly elongated spindle [18, 39, 40]. Treatment with A23187 in combination with 6-DMAP or CHX resulted in pronuclear development, as did IVF (90–100%). However, one pronucleus without second polar body extrusion was observed at 4 h in the A23187+6-DMAP group, while nuclear formation was not detected until 8 h after treatment in the majority of A23187+CHX-treated and IVF oocytes. CD treatment inhibited the second polar body extrusion in A23187+CHX-treated oocytes, which may lead to diploid development.
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Inactivation of MPF and MAPK Following Parthenogenetic Activation and IVF
As similar progression of pronuclear development was observed in A23187+CHX-treated and IVF oocytes but quite different progression in A23187+6-DMAP-treated oocytes, the H1 and MAP kinase activities were analyzed following treatments with A23187+DMAP and IVF (Figs. 1 and 2). A23187+6-DMAP treatment caused a significant reduction of H1 kinase activity by 30 min, and the value subsequently remained low. MAPK activity, on the other hand, began to decrease at 3 h and reached its nadir at 15 h. Inactivation of MPF preceded inactivation of MAPK (Fig. 1). In IVF oocytes (Fig. 2), inactivation of MPF also preceded inactivation of MAPK. Sperm penetration, estimated to occur at 2 h after sperm-oocyte coincubation, induced instant inactivation of H1 kinase at 3–4 h after the start of IVF, and the activity stayed low (10–20%) during the remaining period. Inactivation of MAPK, however, occurred at 10 h (around 60% reduction) after sperm-oocyte coincubation and then reached basal levels at 30–40% of activity at 12–18 h after IVF.
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Cell Cycle Protein Changes Following Parthenogenetic Activation and IVF
As a continuation of the MPF and MAPK assay experiment, the constituent proteins of these kinases were analyzed. In metaphase II oocytes, the accumulation of cyclin B1, cdc2, and ERK2 was prominent (Figs. 3 and 4, A and B). In oocytes treated with A23187 alone, cyclin B1 was destroyed 1 h after treatment and then increased from 4 h to 15 h. However, there was no change in cdc2 and ERK2 at these time points. In oocytes treated with A23187+6-DMAP, the reduction of cyclin B1 was observed at 1 h and the accumulation of B1 at 4 and 15 h. Band shift in cdc2, indicating phosphorylation, occurred at 4 and 15 h after the combined treatment but not after treatment with A23187 alone (compare bands C and D in Fig. 3). Band shift in ERK2, indicating dephosphorylation, began at 4 h and was complete by 15 h (Fig. 4B). In oocytes treated with A23187+CHX, the protein changes differed from those in A23187+6-DMAP-treated oocytes. While the reduction of cyclin B1 was observed at 1 h after treatment with A23187+CHX, no increases were observed until 15 h. Band shift in cdc2 and ERK2 occurred not at 4 h but at 15 h. In the IVF oocytes, patterns of protein change were similar to those in A23187+CHX-treated oocytes, except for a 2-h delay for the IVF group due to the time needed for sperm penetration. Accumulation of cdc25 was not detected in metaphase II oocytes and oocytes treated with A23187 alone or with A23187+CHX during the observation period (Fig. 4C). However, cdc25 was detected at 15 h in oocytes treated with A23187+6-DMAP and at 24 h in fertilized oocytes.
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Microinjection of ERK2 Antibody into Oocytes
In order to clarify whether pronuclear formation was indeed caused by inactivation of MAPK, anti-ERK2 was directly injected into A23187-treated oocytes. For each microinjection batch, matured oocytes at 24 h with one polar body were selected and distributed into three groups. In the group treated with A23187 alone, an average of 65% oocytes (n = 87) were activated (metaphase III chromosomes and pronuclear formation), and 11% with pronuclear formation were observed (Fig. 5, control). In the oocytes treated with A23187 followed 4 h later by microinjection of anti-ERK2, 70% (n = 98) were activated and 43% oocytes formed pronuclei. By contrast, only 11% (n = 86) of control oocytes microinjected with buffer solution after treatment with A23187 formed pronuclei (p < 0.01). Therefore, ERK2 antibody, not the injection procedure, induced pronuclear formation in oocytes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In mammalian oocytes arrested at metaphase II, cyclin degradation is either inhibited (as in Xenopus, [42]) or slowed down, and the levels are maintained by new synthesis of cyclin B (as in mice; [43]). Mos, a component of cytostatic factor (CSF), may arrest eggs at metaphase II by maintaining high MPF activity via the prevention of cyclin degradation, while the MAPK pathway may mediate the function of Mos [44, 45]. Previous work clearly demonstrated the necessity of MAPK for CSF activity in mammals [12, 46]. Mos/MAPK pathway may cause metaphase arrest by its action on other proteins such as CENP-E that are essential for the metaphase-anaphase transition [47]. In all mammalian species studied so far, fertilization triggers a transient increase in cytoplasmic free Ca2+, which brings about inactivation of CSF and MPF [28, 48]. Inactivation of MPF is associated with cyclin degradation and is closely linked to the meiotic release ([42], present study in cattle). The present results suggested further that initial inactivation of MPF was clearly due to decreased amounts of cyclin but not related to cdc2 kinase. The low MPF activity after either artificial activation or fertilization appeared to be maintained by phosphorylation of cdc2, which occurred in 6-DMAP and IVF- or CHX-treated oocytes. The differences are that earlier phosphorylation of cdc2 was detected in 6-DMAP-treated oocytes while later phosphorylation of cdc2 was detected in IVF- or CHX-treated oocytes. This may relate to the different timing in pronuclear progression after different treatments. For inactivation of MAPK, in the A23187+6-DMAP treatment group, earlier dephosphorylation of ERK2 again correlated with earlier inactivation of MAPK. However, in IVF- and A23187+CHX-treated oocytes, dephosphorylation of ERK2 was not observed at 4 h, but at 15 h when a pronucleus was found. Although A23187 alone could not induce pronuclear development, the first calcium stimulation seemed to be very important for further development, since 6-DMAP alone could induce morphological changes similar to those in A23187+6-DMAP-treated oocytes whereas further development in oocytes treated with 6-DMAP alone was limited [4, 18]. In agreement with Moos et al. [49], we demonstrated that CHX treatment induced temporal changes in the decrease in both MPF and MAPK in matured bovine oocytes similar to those occurring after fertilization. CHX was also shown to inhibit cyclin B1 synthesis during bovine oocyte maturation [50]. It seems that both the first calcium increase and the subsequent inhibition of certain kinases or proteins by artificial activation protocols are indispensable for normal progression of the first mitotic cell cycle.
Cyclin B1 protein levels were high in the unfertilized oocyte, declined upon fertilization or artificial activation, and reaccumulated to a high level during the first cell cycle. Anaphase and cyclin degradation are mediated by the same destruction box-dependent machinery, but may be independent events [51]. MPF must be inactivated for the cell to complete cytokinesis and return to the interphase; p34cdc2 is phosphorylated on Thr-14 and Tyr-15 to prevent the premature activation of p34cdc2/cyclin B [52]. Active MPF is composed of a certain amount of cyclin B and dephosphorylation state on Tyr15 and Thr14 and phosphorylation on Thr161 of cdc2 [51]. cdc25 initiates mitosis by dephosphorylating cdc2 on Tyr15 and probably on Thr14, thus activating MPF. cdc25B is an unstable activator of p34cdc2/cyclin B, therefore functioning as a trigger for mitosis; it is a short-life protein [53]. This protein can be detected only at a certain time before mitotic entry. In this experiment, cdc25 was observed at 15 h in A23187+6-DMAP-treated oocytes and at 24 h in IVF oocytes. Furthermore, the accumulation of cyclin B1 and dephosphorylation of cdc2 occurred before the first mitotic entry in bovine oocytes. Therefore, the accumulation of cyclins and the phosphorylation state of cdc2 are important in determining the timing of entry into mitosis.
It has been clearly shown that activation of MAPK/ERK2 is caused by dual phosphorylation, and the mechanism has been elucidated [54]. We compared the kinetics of pronuclear development with the activity of MAPK following treatments. In parallel, to detect phosphorylated MAPK through shift in electrophoretic mobility, cell lysates were examined by immunoblot analysis with anti-p42MAPK antibody. Alternatively, its activity was measured by phosphorylation status in an in-gel kinase assay [22], in which the MAP activity was assayed by analyzing the reaction with its substrate myelin basic protein. Both assays revealed that p42MAPK is fully activated in mature oocytes, while the faster-migrating, inactive form of p42MAPK begins to appear 3–4 h after A23187+6-DMAP treatments. Therefore, the time at which MAPK begins to become dephosphorylated coincides with the appearance of pronuclear formation. A physiological MAPK phosphatase, named MKP-1, was found to inactivate MAPK [55].
MAPK inactivation is necessary for oocyte activation and pronuclear development. Further evidence in starfish oocytes showed that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development [56]. In pronuclear oocytes, cdc2 is phosphorylated while ERK2 is dephosphorylated. During meiotic maturation of Xenopus oocytes, cdc2 is tyrosine dephosphorylated whereas p42MAPK becomes tyrosine phosphorylated at the time of GVBD [57]. Therefore, the balance of phosphate seems to play an important role in the activation MPF and MAPK. The 6-DMAP was also found to inhibit protein phosphorylation and GVBD in mouse and bovine oocytes [58, 59]. As 6-DMAP causes dephosphorylation of ERK2 but phosphorylation of cdc2 either directly or indirectly, it might be inappropriate to consider 6-DMAP as a phosphorylation inhibitor in general.
The general model for cell cycle regulation in bovine oocyte activation can be summarized as shown in Figure 6. Metaphase II-arrested oocytes are characterized by high levels of MPF and MAPK activities, corresponding with high levels of cyclin B, dephosphorylation status of cdc2, and phosphorylation of ERK2. Cyclin B degradation is responsible for the instant inactivation of MPF activity. Low MPF activity can be maintained by either inhibition of continuous synthesis of cyclin B (CHX) or by phosphorylation of cdc2, which may cause or be caused by dephosphorylation of ERK2 (6-DMAP). Inactivation of MPF is parallel to meiotic release, and inactivation of MAPK is associated with pronuclear development. Before mitotic entry, accumulation of cdc25 is important for dephosphorylation of cdc2. In contrast to CHX- or IVF-induced activation, 6-DMAP induces earlier dephosphorylation of MAPK and therefore earlier pronuclear development. The reactivation of MPF before mitotic entry is induced by accumulation of cyclin B and dephosphorylation of cdc2. In partial activation (metaphase III) induced by calcium ionophore A23187 alone, only cyclin B destruction, but no change in cdc2, cdc25, and ERK2, was observed. In full activation (pronuclear development) induced by A23187+6-DMAP, both cyclin B destruction and phosphorylation of cdc2, dephosphorylation of ERK2, and later cdc25 accumulation occurred. This argues for the proposition that the first calcium transient is for the destruction of cyclin B while later calcium oscillations trigger calcium-dependent proteolytic pathway, leading to the destruction of continuously newly synthesized cyclin B. Later calcium oscillations during fertilization may also involve phosphorylation-dependent kinase pathways. MPF inactivation is just a starter; MAPK inactivation determines whether oocytes undergo further pronuclear development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: X. Yang, Department of Animal Science, 3636 Horsebarn Road Ext 40, University of Connecticut, Storrs, CT 06269. FAX: 860 486 4375; xyang{at}ansc1.cag.uconn.edu
3 Current address: Laboratory of Reproductive Medicine, Brown University, Women & Infants Hospital, Marine Biological Laboratories, Lillie 321, 7 MBL Street, Woods Hole, MA 02540.
Accepted: February 2, 1999.
Received: November 9, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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