BIOLOGY OF REPRODUCTION 59, 537–545 (1998)
Differential Inactivation of Maturation-Promoting Factor and Mitogen-Activated
Protein Kinase Following Parthenogenetic Activation of Bovine Oocytes1
Lin Liu, Jyh-Cherng Ju, and Xiangzhong Yang2
Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269
ABSTRACT
ine/threonine kinases, which require phosphorylation to become activated [9]. The precise changes in the activity of
MAPK and its relation to MPF during oocyte activation,
however, remain unclear in most mammalian species.
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([21], our unpublished results).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 calciumelevation 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.
Bovine oocytes matured for 24 h (young) or 40 h (aged) were
treated with calcium ionophore (A23187) alone or followed
with 6-dimethylaminopurine (6-DMAP), a protein phosphorylation inhibitor, and were then assayed for histone H1 kinase and
mitogen-activated protein kinase (MAPK) activities. Additionally,
the changes in chromatin, meiotic spindle, and microfilament
were assessed by immunofluoresence microscopy. In both young
and aged oocytes, treatment with 6-DMAP following A23187
treatment abolished the activities of both H1 and MAPKs; the
decline of H1 kinase preceded the decline in MAPK activity.
However, A23187 treatment alone caused a slower decrease in
H1 kinase activity and no evident MAPK alteration in young
oocytes. In contrast, activities of both kinases decreased in aged
oocytes after A23187 treatment, similar to the response in the
combined treatments. The inactivation of MAPK was caused by
dephosphorylation of MAP42/extracellular signal-regulated kinase 2 (ERK2) as detected by gel mobility shift in the Western
blot assay. A23187 treatment of young oocytes led to chromosome separation and second polar body extrusion, but not pronuclear development, with the majority of the oocytes arrested
at a transitional stage of metaphase to anaphase known as metaphase III (MIII). However, most of the A23187-treated aged oocytes developed to the pronuclear stage. When oocytes, regardless of age, were treated by A23187 plus 6-DMAP, bivalent chromosomes were clumped into a single mass, the spindle was disassembled, microtubule networks were distributed in the
cytoplasm, and a pronucleus appeared. It is suggested that the
decrease in H1 kinase activity is involved in the initiation of
oocyte activation, i.e., the exit from metaphase II, whereas the
decrease in MAPK activity correlates with onset of pronuclear
formation. In conclusion, inactivation of maturation-promoting
factor and MAPKs probably occurs via two independent processes, and the inactivation of both kinases is required for the
metaphase II oocytes to progress through interphase. High
MAPK activity might contribute to spindle stabilization, and inactivation of MAPK is associated with microtubular network formation in the cytoplasm.
INTRODUCTION
The increase in maturation-promoting factor (MPF), as
well as mitogen-activated protein kinase (MAPK) activities,
was found to be necessary for the onset of germinal vesicle
breakdown and metaphase progression during oocyte maturation and meiotic arrest [1–5]. MPF is composed of cyclin B and p34cdc2 kinase, and displays a cyclic activity that
peaks at metaphase [6]. The reduction of histone H1 kinase
is an indicator of MPF inactivation [6–8]. MAPKs are serAccepted April 14, 1998.
Received January 21, 1998.
1
This work was supported in part by the Cooperative State Research,
Education, and Extension Service, U.S. Department of Agriculture, under
Agreement No. 96–35203–3268. This is a scientific contribution (number
1793) of the Storrs Agricultural Experiment Station of the University of
Connecticut.
2
Correspondence. FAX: (860) 486–4375;
e-mail: [email protected]
537
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LIU ET AL.
MATERIALS AND METHODS
Chemicals and Hormones
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 24–278C. 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 mg/ml ovine FSH,
5.0 mg/ml ovine LH, and 1.0 mg/ml estradiol). For maturation culture, cumulus-enclosed oocytes were cultured in
100-ml droplets of maturation medium (20–25 oocytes/
droplet) covered with mineral oil at 398C in 5% CO2 and
humidified air.
from culture, transferred to 4 ml cold (48C) collection buffer
(6.4 mM EDTA, 10 mM NaFl, and 100 mM Na3VO4 in
PBS) in Eppendorf tubes, and stored frozen at 2708C. During the kinase assay, the tubes were placed on ice, and 6
ml of homogeneous buffer (HB) was added to each sample
(HB: 45 mM b-glycerophosphate, 12 mM p-nitrophenylphosphate, 20 mM 3-[N-morpholino]-propanesulfonic acid,
pH 7.2, 12 mM MgCl2, 12 mM ethylene glycol-bis (baminoethylether) N,N,N1,N1-tetra-acetic acid, 0.8 mM dithiothreitol, 0.1 mM Na3VO4, 20 mg/ml leupetin, and 40
mg/ml aprotinin). The samples were then incubated at 378C
for 15 min. After incubation, 8 ml of kinase buffer (KB)
was added (KB: HB with 2 mg/ml histone H1 and 1 mg/
ml MBP, 1.2 mM protein kinase inhibitor, and 250 mCi/ml
[g-32P]ATP [Dupont, Boston, MA]). Samples were incubated for an additional 30 min at 378C, and the reaction
was terminated by the addition of 18 ml double-strength
SDS sample buffer [30]. The samples were heated at 958C
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 xray 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.
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 mM calcium ionophore (A23187)
either alone or followed by incubation with 2.5 mM 6DMAP for 4 h. Treated oocytes were washed and then cultured in 100-ml drops of modified KSOM [27,28] containing 0.1% BSA and 0.4 mM taurine under mineral oil at
398C 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 (2708C) 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
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 SDSPAGE and transferred onto a polyvinylidene fluoride membrane (PVDF; Immobilon-P; Millipore, Bedford, MA). The
membrane was blocked with 5% nonfat dry milk in Trisbuffered 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
378C in a microtubule-stabilizing buffer [31]. The oocytes
were washed extensively and blocked overnight at 48C 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 a- and b-tubulin mouse monoclonal anti-
KINASE ACTIVITIES OF ACTIVATED BOVINE OOCYTES
539
FIG. 1. Autoradiogram of typical changes
of histone H1 and MAPK activities in activated young (A) and aged (B) bovine oocytes as demonstrated by phosphorylation
of histone H1 and MBP. In control oocytes, oocytes were not treated with
chemicals but cultured for the same period. C, Calcium ionophore A23187 treatment; D, A23187 1 6-DMAP treatment;
M, matured oocytes as controls.
bodies (1:200), washed, and then incubated with fluorescein
isothiocyanate (FITC)-conjugated anti-mouse IgG (1:200;
Cappel, West Chester, PA) at 378C 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 mg/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.
RESULTS
To determine the kinetics of the inactivation of MPF and
MAPKs, we collected samples of activation-treated oocytes
and assayed them for their capacity to phosphorylate MBP
(MAPK) and histone H1 (MPF). The typical changes in
both kinase activities are shown in Figure 1. In young oocytes, histone H1 kinase activity decreased abruptly when
oocytes were treated by A23187 plus 6-DMAP, while it
decreased more slowly when they were treated by A23187
alone. In control oocytes without any treatments, H1 kinase
activity declined gradually but remained at relatively high
levels compared to those of activated oocytes. MAPK activity, on the other hand, remained high in the A23187treated oocytes as well as in the metaphase control oocytes.
However, MAPK activity decreased between 3 and 4 h after
treatment with A23187 plus 6-DMAP and remained at very
low levels at 15 h. To determine whether the inactivation
of MAPK was due to the dephosphorylation of the kinase,
the mobility shift of MAP42/ERK2 was analyzed by Western blot analysis. Figure 2 shows that a mobility shift was
observed at 4 and 15 h in young oocytes treated by A23187
plus 6-DMAP but no shift was observed with A23187 treatment alone. The ERK2 band shift to a faster-migrating form
coincided with the time of MAPK inactivation. Furthermore, when oocytes were treated with A23187 alone, no
mobility shift was observed and MAPK was not inactivated
(Figs. 1 and 2).
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.
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]).
A23187 treatment alone. A23187 treatment of newly
matured oocytes (24-h IVM) induced the extrusion of a
FIG. 2. Dephosphorylation of MAPKs (ERK2) as detected by mobility
shift in young bovine oocytes after treatment with calcium ionophore
A23187 alone (C) or A23187 1 6-DMAP (D) shown by Western blotting.
M, Matured oocytes (24-h IVM). Fifty oocytes in each lane were used.
The results are representative of two independent experiments.
540
LIU ET AL.
FIG. 3. Changes in histone H1 and MAPK activities in young (A) and
aged (B) oocytes following treatment with A23187 alone. Experiments
were repeated three times with similar results. Average values are shown.
a, significant difference (p , 0.05) in histone H1 kinase activity between
treated oocytes and their respective controls; b, significant difference (p
, 0.05) in MAPK activity between treated oocytes and their respective
controls.
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 5 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
FIG. 4. Changes in histone H1 and MAPK activities in young (A) and
aged (B) oocytes after treatment with A23187 1 6-DMAP. Experiments
were repeated three times with similar results. Average values are shown.
a, significant difference (p , 0.05) in histone H1 kinase activity between
treated oocytes and their respective controls; b, significant difference (p
, 0.05) in MAPK activity between treated oocytes and their respective
controls.
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 15h 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
KINASE ACTIVITIES OF ACTIVATED BOVINE OOCYTES
541
FIG. 5. Nuclear and microtubule behavior in young bovine oocytes after activation treatments. A) A 24-h IVM oocyte. B) A 28-h IVM oocyte. C–F)
Oocytes after treatment with A23187 alone at 1 hr (C, early anaphase), at 3–4 h (D, polar body extrusion; E, spindle separation), and at 15 h (F, MIII
chromosomes and spindle). G–K) Oocytes after treatment with A23187 1 6-DMAP: at 0.5 h (G, chromosome aggregation and spindle destruction), at
3–4 h (H, chromosome decondensation; I, microtubular network), and at 15 h (J, one pronucleus and one polar body; K, microtubular network). L)
Aged oocytes (24-h IVM plus 15-h culture in KSOM). Blue: Hoechst-stained for DNA; green: FITC-stained for tubulin; red: rhodamine-stained for actin
microfilament. PB, Polar body; PN, pronucleus; CH, chromosomes; MT, microtubules; SP, spindle. Bar 5 35 mm.
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 5 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 5 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.
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),
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LIU ET AL.
FIG. 6. Nuclear and microtubule behavior in aged bovine oocytes after activation treatments. A) A 40-h IVM oocyte (oocytes freed of cumulus cells
at 24-h IVM followed by culture in KSOM for 16 h). B–G) Oocytes after treatment with A23187 alone: at 0.5 h (B, late anaphase; C, early anaphase),
at 1 h (D, telophase; E, spindle), at 3–4 h (F, polar body extrusion), and at 15 h (G, one pronucleus and two polar bodies). H–L) Oocytes after treatment
with A23187 1 6-DMAP: at 0.5 h (H, chromosome aggregation; I, spindle destruction), at 3 h (J, pronucleus; K, microtubular network), and at 15 h
(L, mitotic phase). Blue: Hoechst-stained for DNA; green: FITC-stained for tubulin; red: rhodamine-stained for actin (microfilament). PB, Polar body;
PN, pronucleus; CH, chromosomes; MT, microtubules; SP, spindle. Bar 5 35 mm.
and 45% (n 5 29) of oocytes were at mitotic metaphase or
anaphase by 15 h after treatment (Fig. 6L).
DISCUSSION
In this study, we examined the dynamics in MAP (MBP)
kinase versus MPF (H1 kinase) activity during bovine oocyte activation. Additionally, morphological changes in microtubule organization and chromatin behavior were observed. In oocytes undergoing full activation (defined as
pronuclear formation), MPF inactivation occurred more
quickly and preceded MAPK inactivation; whereas in oocytes undergoing partial activation (defined as exit from
MII arrest but no pronuclear formation), MPF was inactivated but MAPK activity remained high. It is clear from
our study that a decrease in MPF activity coincided with
MII exit and that a decrease in MAPK activity coincided
with pronuclear formation. Therefore, the MAPK inactivation was independent of MPF inactivation. This observation is supported by previous reports that the decline in
MPF activity was also found to precede the decrease in
MAPK activity in mouse and Xenopus oocytes [10, 12, 13].
Two additional lines of evidence supporting our conclusion
of independent inactivation of the two kinases are that
MAPK activity remained high in young oocytes treated by
A23187 alone, while MPF was inactivated, and that MAPK
activity declined long after MPF activity declined in aged
oocytes treated by this ionophore alone.
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 main-
KINASE ACTIVITIES OF ACTIVATED BOVINE OOCYTES
tained 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 MIto-MII transition in both bovine and mouse oocytes [1, 3].
The incomplete activation of oocytes, resulting in an MIIto-MIII transition (MIII arrest), is very similar to the MIto-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
543
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 6DMAP, 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-DMAPdependent 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
544
LIU ET AL.
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.
ACKNOWLEDGMENTS
The authors wish to thank M. Julian for her help with revising this
manuscript, and X. Tian, J. Xu, S. Jiang, B. Jeong, C. Xu, H. Freake, and
M. MacGrane for their help with the experiments. Ovine FSH and LH
used throughout our research were kindly provided by 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.
REFERENCES
1. Velhac MH, de Pennart H, Maro B, Cobb MH, Clarke HJ. MAP
kinase becomes stably activated at metaphase and is associated with
microtubule-organizing centers during meiotic maturation of mouse
oocytes. Dev Biol 1993; 158:330–340.
2. Chesnel F, Eppig JJ. Induction of precocious germinal vesicle breakdown (GVB) by GVB-incompetent mouse oocytes: possible role of
mitogen-activated protein kinases rather than p34cdc2 kinase. Biol
Reprod 1995; 52:895–902.
3. Fissore RA, He CL, Vande Woude GF. Potential role of mitogenactivated protein kinase during meiosis resumption in bovine oocytes.
Biol Reprod 1996; 55:1261–1270.
4. Matten WT, Copeland TD, Ahn NG, Vande Woude GF. Positive feedback between MAP kinase and Mos during Xenopus oocyte maturation. Dev Biol 1996; 179:485–492.
5. Wu B, Ignotz G, Currie B, Yang X. Dynamics of maturation-promoting factor and its constituent proteins during in vitro maturation of
bovine oocytes. Biol Reprod 1997; 56:253–259.
6. Nurse P. Universal control mechanism regulating onset of M-phase.
Nature 1990; 344:503–508.
7. Labbé J, Picard A, Peaucellier G, Cavadore JC, Nurse P, Dorre M.
Purification of MPF from starfish: identification as the histone H1
kinase p34cdc2 and a possible mechanism for its periodic activation.
Cell 1989; 57:253–263.
8. Moos J, Kopf GS, Schultz RM. Cycloheximide-induced activation of
mouse oocytes: effects on cdc2/cyclin B and MAP kinase activities.
J Cell Sci 1996; 109:739–748.
9. Posada J, Cooper JA. Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes. Science 1992; 255:212–215.
10. Ferrell JE, Wu M, Gerhart JC, Martin GS. Cell cycle tyrosine phosphorylation of p34 cdc2 and microtubule associated protein kinase homolog in Xenopus oocytes and eggs. Mol Cell Biol 1991; 11:1965–
1971.
11. Collas P, Sullvian EJ, Barnes FL. Histone H1 kinase activity in bovine
oocytes following calcium stimulation. Mol Reprod Dev 1993; 34:
224–231.
12. Lorca T, Cruzalegui FH, Fesquet D, Cavadore JC, Mery J, Means A,
Doree M. Calmodulin-dependent protein kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs. Nature
1993; 366:270–273.
13. Velhac MH, Kubiak JZ, Clarke HJ, Maro B. Microtubule and chro-
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
matin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes. Development 1994; 120:1017–1025.
Moos J, Visconti PE, Moore GD, Schultz RM, Kopf GS. Potential
role of mitogen-activated protein kinase in pronuclear envelope assembly and disassembly following fertilization of mouse eggs. Biol
Reprod 1995; 53:692–699.
Moos J, Xu Z, Schultz RM, Kopf GS. Regulation of nuclear envelope
assembly/disassembly by MAP kinase. Dev Biol 1996; 175:358–361.
Sagata N. What does Mos do in oocytes and somatic cells? Bioessays
1997; 19:13–21.
Liu L, Ju JC, Yang X. Parthenogenetic development and protein patterns of newly matured bovine oocytes following chemical activation.
Mol Reprod Dev 1998; 49:298–307.
Soloy E, Kanka J, Viuff D, Smith SD, Callesen H, Greve T. Time
course of pronuclear deoxyribonucleic acid synthesis in parthenogenetically activated bovine oocytes. Biol Reprod 1997; 57:27–35.
Swann K, Lai FA. A novel signaling mechanism for generating Ca21
oscillations at fertilization in mammals. Bioessays 1997; 19:371–378.
Susko-Parrish JL, Liebfried-Rutledge ML, Northey DL, Schutzkus V,
First NL. Inhibition of protein kinases after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without
meiotic completion. Dev Biol 1994; 166:729–739.
Uranga JA, Pedersen RA, Arechaga J. Parthenogenetic activation of
mouse oocytes using calcium ionophores and protein kinase C stimulators. Int J Dev Biol 1996; 40:515–519.
Szollosi MS, Kubiak JZ, Debey P, de Pennart H, Szollosi D, Maro B.
Inhibition of protein kinases by 6-dimethylaminopurine accelerates the
transition to interphase in activated mouse oocytes. J Cell Sci 1993;
104:861–872.
Moses RM, Masui Y. Enhancement of mouse egg activation by the
kinase inhibitor, 6-dimethylaminopurine (6-DMAP). J Exp Zool 1994;
270:211–218.
Moses RM, Kline D, Masui Y. Maintenance of metaphase in colcemid-treated mouse eggs by distinct calcium- and 6-dimethylaminopurine
(6-DMAP)-sensitive mechanisms. Dev Biol 1995; 167:329–337.
Yang X, Jiang S, Foote RH. Bovine oocyte development following
different oocyte maturation and sperm capacitation procedures. Mol
Reprod Dev 1993; 34:94–100.
Presicce GA, Yang X. Nuclear dynamics of parthenogenesis of bovine
oocytes matured in vitro for 20 and 40 hours and activated with combined ethanol and cycloheximide treatment. Mol Reprod Dev 1994;
37:61–68.
Lawitts JA, Biggers JD. Culture of preimplantation embryos. In: Wassarman PM, DePamphilis ML (eds.), Guide to Techniques in Mouse
Development, Methods in Enzymology. San Diego: Academic Press;
1993: 153–164.
Liu Z, Foote RH. Effects of amino acids on the development of invitro matured in-vitro fertilized bovine embryos in a simple proteinfree medium. Hum Reprod 1995; 10:2985–2991.
Christmann L, Jung T, Moor RM. MPF components and meiotic competence in growing pig oocytes. Mol Reprod Dev 1994; 38:85–90.
Laemmli UK. Cleavage of structural proteins during assembly of the
head of bacteriophage T4. Nature 1970; 227:680–685.
Allworth AE, Albertini DF. Meiotic maturation in cultured bovine oocytes is accompanied by remodeling of the cumulus cell cytoskeleton.
Dev Biol 1993; 158:101–112.
Kubiak JZ. Mouse oocytes gradually develop the capacity for activation during the metaphase II arrest. Dev Biol 1989; 136:537–545.
Sagata N, Watanabe N, Vande Woude GF, Ikawa Y. The c-mos protooncogene product is a cytostatic factor responsible for meiotic arrest
in vertebrate eggs. Nature 1989; 342:512–518.
Watanabe N, Hunt T, Ikawa Y, Sagata N. Independent inactivation of
MPF and cytostatic factors (mos) upon fertilization of Xenopus eggs.
Nature 1991; 352:247–248.
Haccard O, Sarcevic B, Lewellyn A, Hartley R, Roy L, Izumi T,
Erikson E, Maller JL. Induction of metaphase arrest in cleaving Xenopus embryos by MAP kinase. Science 1993; 262:1262–1265.
Velhac MH, Kubiak JZ, Weber M, Geraud G, Colledge WH, Evans
MJ, Maro B. Mos is required for MAP kinase activation and is involved in microtubule organization during meiotic maturation in the
mouse. Development 1996; 122:815–822.
Nebreda AR, Hunt T. The c-mos proto-oncogene protein kinase turns
on and maintains the activity of MAP kinase, but not MPF, in cellfree extract of Xenopus oocytes and eggs. EMBO J 1993; 12:1979–
1986.
KINASE ACTIVITIES OF ACTIVATED BOVINE OOCYTES
38. Thibier C, De Smedt V, Poulhe R, Huchon D, Jesus C, Ozon R. In
vitro regulation of cytostatic activity in Xenopus metaphase II-arrested
oocytes. Dev Biol 1997; 185:55–66.
39. Roy LM, Haccard O, Izumi T, Lattes BG, Lewellyn AL, Maller JL.
Mos-proto-oncogene function during oocyte maturation in Xenopus.
Oncogene 1996; 12:2203–2211.
40. Shibuya EK, Boulton TG, Cobb MH, Ruderman JV. Activation of
p42 MAP kinase and the release of oocytes from the cell cycle arrest.
EMBO J 1992; 11:3963–3975.
41. Takenaka K, Gotoh Y, Nishida E. MAP kinase is required for the
spindle assembly checkpoint but is dispensable for the normal M
phase entry and exit in Xenopus egg cell cycle extracts. J Cell Biol
1997; 136:1091–1097.
42. Peter M, Nakagawa J, Doree M, Labbe JC, Nigg EA. In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation
of lamins by cdc2 kinase. Cell 1990; 61:591–602.
43. Heald R, Mckeon F. Mutations of phosphorylation sites in lamin A
44.
45.
46.
47.
48.
49.
545
that prevent nuclear lamina disassembly in mitosis. Cell 1990; 61:
579–589.
Burke B, Gerace L. A cell free system to study reassembly of the
nuclear envelope at the end of mitosis. Cell 1986; 44:639–652.
Kubiak JZ, Weber M, Geraud G, Maro B. Cell cycle modification
during the transitions between meiotic M-phases in mouse oocytes. J
Cell Sci 1992; 102:457–467.
Wang XM, Zhai Y, Ferrell JE Jr. A role for mitogen-activated protein
kinase in the spindle assembly checkpoint in XTC cells. J Cell Biol
1997; 137:433–443.
Vincent C, Cheek TR, Johnson MH. Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent
on the level of internal calcium. J Cell Sci 1992; 103:389–396.
Winston NJ, McGuiness O, Johnson MH, Maro B. The exit of mouse
oocytes from meiotic M-phase requires intact spindle during intracellular calcium release. J Cell Sci 1995; 108:143–151.
Collas P, Chang T, Long C, Robl JM. Inactivation of histone H1 kinase
by Ca21 in rabbit oocytes. Mol Reprod Dev 1995; 40:253–258.