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GREAT EXPERIMENTS
The discovery of MPF
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Yoshio Masui
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Before dividing, eukaryotic cells undergo
a highly ordered series of events called the cell cycle. These events
include S phase, when the cell's DNA is replicated, and mitosis (M
phase), when the replicated chromosomes are separated. These phases are
separated by periods called gap (G) phases, with the G1 phase preceding
S phase and the G2 phase occurring between S phase and mitosis. How the
cell controls this essential series of events is one of the fundamental
problems of cell biology.
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One prominent early approach to studying
control of the cell cycle was to introduce a nucleus from one phase of
the cell cycle into a cytoplasm from another. Techniques for making
such nucleo-cytoplasmic hybrid cells, including nuclear transplantation
and cell fusion, became available in the 1950s, but it was not until
the late 1960s that they were used to study cell cycle activities such
as the initiation of DNA synthesis and the condensation of chromosomes.
This type of experiment included nuclear transplantation by injection
in frog oocytes and eggs (1577), excision and transplantation
of cytoplasmic fragments in protozoa (1575), and virus-mediated
fusion between tissue culture cells (1590).
In all cases the nucleus conformed to the cell cycle stage of the
cytoplasm, indicating that cytoplasmic factors control nuclear
activities during the cell cycle. Evidence for the existence of factors
which might control the initiation of cell cycle events came from other
types of experiments. For example, experiments done with Tetrahymena
showed that heat shock synchronized the cell cycles of a population of
cells, presumably because of the heat lability of a component that
promotes cell division (1583).
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Despite what these experiments revealed
about the organization of the cell cycle, none of them provided any
clue to the identity of the factors responsible for cytoplasmic control
over the nucleus. Nor could any of the experiments be readily adapted
to provide an assay with which to identify those factors and study
their biochemical mechanisms. That would require an experimental system
in which cytoplasm from particular stages of the cell cycle could be
isolated and used to cause a transition from one phase of the cell
cycle to another. The frog oocyte provided this system and played an
essential role in identifying the molecular machinery that drives the
cell cycle.
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Background
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Until the early 1960s, I worked on the
problem of embryonic induction and was interested in the mechanism of
cell differentiation. Markert's hypothesis of differential gene
activation in development, published in 1958 (1584), greatly
interested me and I joined his lab at Yale University on my sabbatical
leave in 1966 to learn his approach to cell differentiation. My first
project was a study of lactate dehydrogenase isoenzymes using frozen
penguin embryos sent by an expeditionary team from the Antarctic. The
results turned out to be so complicated that I felt pessimistic about
continuing to study cell differentiation. At the time, similarly
complicated results in other systems had caused many embryologists to
lose confidence in discovering a specific inducer of cell
differentiation. Even the nature of cell differentiation was in some
doubt. Recent results in immunology had raised the possibility that
differentiation might be a clonal selection process rather than a cell
transformation caused by the inducer. To escape such scepticism, I
wanted to study a well-defined developmental change inducible in a
single cell by a highly specific inducer. Maturation of frog oocytes
seemed to fit the bill.
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Figure 1
The events of maturation and early development.
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Frog oocytes undergo a long period of growth within
the follicles of the ovary before "maturing" into unfertilized eggs. Figure 1
shows that fully grown oocytes are naturally arrested in the G2 phase
of the cell cycle, immediately preceding the first meiotic division.
Maturation is the term used to describe their progression from that
point through the rest of meiosis; thus, "maturation" is synonymous
with the release of the oocytes from their arrest and their performance
of the divisions of mitosis. The process is initiated in response to
stimulation of the ovary by gonadotropin, a small peptide hormone
secreted by the pituitary. The hormone causes the greatly enlarged
oocyte nucleus (called the "germinal vesicle") to break down and its
chromosomes to condense, and the oocyte then enters and completes the
first meiotic division, passes through prophase of the second, and
finally arrests as an unfertilized egg at metaphase of the second
division. Fertilization relieves this arrest and the egg begins
dividing mitotically. This system suited my needs well, since an
external signal could reliably cause a single cell to undergo a series
of events that was a form of differentiation. Before I began my study
in 1967, however, only a few studies of oocyte maturation had been
published (for review see 1576), and we knew little about how
the signal that acts on the oocyte triggers the events of maturation.
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Most important for my study was a set of experiments by Dettlaff and her colleagues (1578).
They reported that isolated oocytes, freed of the follicle cells which
surround them in the ovary, could be induced to mature by treatment
with gonadotropin. Maturation could also be induced by injecting the
nuclear contents of a maturing oocyte into a second oocyte which had
not been treated with the hormone. These results suggested that the
hormone caused the production of something within the nucleus that was
responsible for initiating maturation. On the basis of the mechanism of
action of other hormones known at the time, they concluded that
gonadotropin probably acted by entering the cell, moving to the
nucleus, and stimulating transcription.
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Seeing these important results, I felt
the need to examine the mode of gonadotropin action on the frog oocyte
further. I first repeated their experiments with Rana pipiens,
the leopard frog, but found that manual removal of follicle tissues
from oocytes was not as easy as they described. I developed a more
reliable way of doing it and found that oocytes completely separated
from follicle cells did not mature in response to gonadotropin, while
those cultured with follicle cells did so (1579). Since it
was known that ovulation induced by gonadotropin could be enhanced by progesterone (1580),
I tried to enhance gonadotropin-induced oocyte maturation by adding
progesterone. However, I found that progesterone alone could induce
follicle-free oocytes to mature (1579). Clearly, it was not
pituitary gonadotropin, but progesterone secreted from follicle cells
stimulated by gonadotropin, that acts on the oocyte to induce its
maturation.
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While I had originally been attracted to
oocyte maturation as a developmental event, I realized that because
maturation is an induced meiotic cell cycle I would also be studying
control of a cell cycle event. Maturation was a highly advantageous
system for this purpose for several reasons. The natural arrest of
immature oocytes in G2, and of unfertilized eggs in M, eliminated the
problems and complicated procedures always involved in synchronizing
the cell cycles of a population of tissue culture cells or
microorganisms, and allowed me to be certain of the cell cycle stage.
In addition, frog oocytes and eggshave the simple advantages of being
big, tough and available in large numbers. As a result it is possible
to inject them and to transfer material from one to another, and to get
large enough quantities of material to do biochemical experiments.
Without these properties my experimental approach would not have been
possible.
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The experiment
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Having identified the real inducer of
maturation as progesterone, I was in a position to ask how it acted
upon the oocyte. First, in order to find the target for progesterone
action, the hormone was injected directly into oocytes up to an
internal concentration of 0.5 µg/ml. However, none of the
progesterone-injected oocytes matured, while oocytes exposed to the
same doses of the hormone in their culture medium did. Therefore, it
was assumed that progesterone could act only on the surface of the
oocyte, and that the hormone must create a signal in the cytoplasm that
acts on the nucleus to initiate maturation (1582).
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Figure 2
A graduated micropipette of the type used to perform cytoplasmic transfer experiments
between oocytes. Each section between two marks holds 5 nl. (Fig. 1 of 1582)
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Figure 3
Maturation-promoting factor activity in the oocyte cytoplasm during maturation and
early development. The horizontal axis indicates the age of the oocyte (hours since
treatment with progesterone) and the vertical axis the concentration of MPF activity
in its cytoplasm. (Fig. 10 of 1582)
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To test this assumption, various volumes of cytoplasm from progesterone-treated
oocytes, measured by the calibrated micropipette shown in Figure 2, were injected
into untreated immature oocytes. These recipient oocytes matured if given more than
5 nl of cytoplasm, as long as the cytoplasm had been removed from the donor oocyte
more than six hours after it had been treated with progesterone. The putative cytoplasmic
substance responsible for this effect was referred to as "maturation promoting factor
(MPF)." It was found that the frequency with which recipient oocytes were induced
to mature increased almost linearly with the volume of the injected cytoplasm. This
allowed measurement of the relative amounts of MPF activity in oocyte cytoplasm
at different times after the initiation of maturation (an amount of MPF was expressed
as the percentage of oocytes induced to mature by injection with a particular volume
of cytoplasm). Figure 3 shows that MPF appears six hours after progesterone treatment
(three hours before breakdown of the nucleus) and persists at a high level until
fertilization, after which it declines to low levels during cleavage (1582).
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Figure 4
Successive transfers of cytoplasm from maturing oocytes to immature oocytes. The
percentage of injected oocytes which matured in a given round is shown.
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If progesterone acting at the surface of the oocyte gives rise to MPF, then MPF
probably appears first in the cytoplasm closest to the surface, without the involvement
of the nucleus. Indeed, MPF was produced when oocytes whose nuclei had been removed
were treated with progesterone. Thus, MPF activity must first be generated in the
cytoplasm and then act upon the nucleus. Since oocytes can be induced to mature
when injected with a volume of cytoplasm equal to less than 1% of their volume,
it was easy to imagine that MPF is amplified in the oocyte cytoplasm by an autocatalytic
reaction. This was demonstrated with a serial transfer experiment shown in Figure
4. An oocyte which had been matured by injection with cytoplasm was itself used
as the donor of cytoplasm for injection into another oocyte. Once it matured, the
second oocyte was used to provide cytoplasm for yet another round of injection and
maturation, and the process continued several more times. When 30 to 40 nl (1%-2%
of an oocyte volume) of cytoplasm were successively transferred in this manner,
no decrease in MPF activity was observed in the cytoplasm of the matured oocytes
at any point in the procedure, despite a dilution of the initial cytoplasm of more
than 100,000 fold in the final oocyte (1582).
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The fact that MPF activity persisted at low levels during the early cleavage divisions
of a fertilized egg (Figure 3) suggested that MPF might promote mitosis as well
as meiosis. If so, injection of MPF-containing cytoplasm from oocytes might be expected
to induce a premature M phase in the cells of a zygote. Unexpectedly, when cytoplasm
from oocytes at late stages of maturation was injected into zygotes, cleavage of
injected blastomeres was arrested at metaphase of the next mitosis, reminiscent
of the arrest of unfertilized eggs at metaphase of the second meiotic division.
This suggested the presence of an inhibitory factor capable of arresting cells in
M phase. The effect of this inhibitory factor, designated "cytostatic factor (CSF),"
showed a dose-dependence similar to that of MPF. It appears shortly after the first
meiotic division and remains until fertilization, after which it quickly disappears.
Using enucleated oocytes it was also found that both production of CSF during oocyte
maturation and its destruction during egg activation could occur independently of
nuclear activities (1582).
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From these experiments we concluded that maturation requires the coordinated activity
of two factors generated in the cytoplasm. MPF appears first, in response to hormonal
stimulation of the oocyte, and drives the oocyte into meiosis. CSF appears with
a delay, perhaps so as not to interfere with the first meiotic division, and seems
responsible for arresting the cell cycle at the following M phase until fertilization.
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The legacy
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Taken together these experiments represented the first quantitative analyses of
cytoplasmic factors that regulate nuclear activities during the cell cycle, and
the first description of a mechanism by which one of them is activated, the autocatalytic
amplification of MPF. However, at that time it was unclear whether MPF is a cell
cycle regulator for both meiosis and mitosis, although we anticipated that this
would be the case because of the presence at low levels of MPF activity in blastomeres
undergoing mitosis. Indeed, MPF was subsequently found to appear shortly before
cells entered mitosis in frog eggs (1585; 1570), cultured mammalian
cells (1571), and yeast cells (1586). This suggested that MPF
is a ubiquitous cell cycle regulator that drives the cell from G2 into mitosis in
all eukaryotes.
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We still had no idea of what MPF is, however. To investigate its chemical nature,
I attempted its extraction from frog eggs. This was unsuccessful until I found that
MPF is quickly inactivated when eggs are homogenized, but remains active in the
liquid fraction when eggs are opened by crushing them with centrifugal force (1568).
Centrifugation of egg extracts through sucrose density gradients showed that MPF
activity sedimented in each of three discrete peaks of 4, 15 and 32S (1568),
the first evidence that MPF activity might reside in a single component (rather
than being a property of a large number of interacting components in the cytoplasm).
RNAases had no effect on MPF activity, but it was quickly inactivated by Ca2+
ions and proteases (1586), and stabilized by ATP and protein phosphatase
inhibitors (1588). These results strongly suggested that MPF contained
at least one phosphoprotein and was dependent on phosphorylation for its activity
(for review see 1576).
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Amazingly, much of the cell cycle would occur in extracts. Nuclei added to extracts
prepared immediately after eggs had been "activated" to mimic fertilization replicated
their DNA, broke down their nuclear envelopes, and formed metaphase chromosomes,
all very much as in vivo(1572). Modified versions of these extracts
could perform up to four complete cell cycles (1573). Simpler extracts
prepared from unfertilized eggs caused the formation of metaphase chromosomes from
the chromatin of added nuclei, consistent with the arrest of the eggs in metaphase.
If, however, the MPF in the extract was inactivated by addition of Ca2+,
nuclei added subsequently behaved as in interphase, proving the important point
that MPF can induce the transition into metaphase (1574).
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A slightly modified form of these extracts, in which nuclei would break down and
condense their chromatin only after MPF was added, provided an assay that allowed
MPF to be purified and characterized in molecular terms (1569). It proved
to be composed of two subunits, cyclin B and cdc2, which together form an active
kinase required for entry into the metaphase states of both the meiotic and mitotic
cell cycles (see The discovery of as the key regulator of the cell cycle).
The kinase itself is regulated by a complicated set of phosphorylations of the cdc2
subunit, and is capable of activating itself through a loop of other protein kinases
and phosphatases. This loop causes a small initial amount of cdc2/cyclinB which
appears at the beginning of the G2/M transition to amplify itself irreversibly,
committing the cell to enter an M phase. This is the basis of the autoamplification
of MPF that we observed in oocyte cytoplasm. Once amplified, MPF phosphorylates
a large number of substrates which then carry out the events of mitosis or meiosis.
The role of MPF, then, is to make the decision when to enter an M phase and to inform
the rest of the cell by phosphorylation (for review see 1587) (and see
M phase kinase regulates entry into mitosis).
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But what about the beginning, both of my own work and of oocyte maturation? How
is MPF initially activated by progesterone to initiate maturation? The hypothesis
that progesterone action is localized to the surface of the frog oocyte was supported
by successful induction of maturation by external application of a polymer-conjugated
progesterone derivative, which cannot enter the cell because of its large size(1576
for review). Nonetheless, neither the exact target of progesterone action nor its
receptor has been identified. Although some of the intermediate steps which connect
progesterone to the production of MPF are now known, such as adenyl cyclase and
PKA inhibition, synthesis of the Mos protein, and activation of MAP kinase, the
precise mechanism of how progesterone acts on the frog oocyte to give rise to MPF
still remains unclear (for review see 1569).
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Acknowledgments
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The author thanks Mrs. Stacey Hayden for her assistance in preparing this article.
The author
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Yoshio Masui was born in Kyoto, Japan in 1931. He graduated from Kyoto University
in 1953 with a B.Sc. in zoology. He earned his M.Sc. (1955) and Ph.D. (1961) from
Kyoto University for his study of the effects of lithium ions on embryonic induction
in amphibians. He taught at the Department of Biology at Konan University, Kobe,
Japan as Lecturer from 1958 and as Assistant Professor from 1965. During a sabbatical
leave in 1966 and 1967 he joined Clement L. Markert's lab at Yale, where he first
studied lactate dehydrogenase isoenzymes in penguin embryos and then initiated his
own study of frog oocyte maturation. In order to complete this study he resigned
his position at Konan University in 1968. He finished the project in 1969, and it
was published in 1971 with Markert as coauthor. After finishing the project in 1969
he moved to Toronto, Canada to assume a position as Associate Professor in the Department
of Zoology, University of Toronto, where he continued his research on cell cycle
regulation using oocytes and early embryos until his retirement in 1997. In 1998,
he was elected a Fellow of the Royal Society, and received the Lasker Basic Medical
Research Award jointly with L. Hartwell, and P. Nurse.
Yoshio Masui
Department of Zoology
University of Toronto
Phone: 416 978 3493
Fax: 416 978 8532
E-mail: masui@zoo.utoronto.ca
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Last Revised on September 10, 2004
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- 1569 Maller, J. L. (1998). Recurring themes
in oocyte maturation. Biol. Cell 90, 453-460. PubMed
- 1575 De Terra, N. (1969). Cytoplasmic control
over the nuclear events of cell reproduction. Int. Rev. Cytol. 25, 1-29.
PubMed
- 1576 Masui, Y. and Clarke, H. J. (1979). Oocyte
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