C Blackwell Wissenschafts-Verlag 2001
Differentiation (2001) 69:1–17
REVIEW
Y. Masui
From oocyte maturation to the in vitro cell cycle:
the history of discoveries of Maturation-Promoting Factor (MPF)
and Cytostatic Factor (CSF)
Accepted in revised form: 3 August 2001
Abstract This article briefly reviews the classical cell
cycle studies using oocytes and zygotes of mainly amphibians in the past century. The discussions are focused
on the investigations into the cytoplasmic factors that
regulate meiosis during oocyte maturation and the
initiation of mitosis during fertilisation, which were carried out in the author’s lab between 1967 and 1987. This
chronicle traces the development of the problems and
the direction in which their solutions were attempted in
the course of these investigations. The author tries to
answer the following questions: why he decided to study
oocyte maturation, how he discovered progesterone as a
maturation-inducing hormone, how he discovered and
characterised the cytoplasmic regulators of the cell cycle,
Maturation-Promoting Factor (MPF) and Cyto-Static
Factor (CSF), and how he invented the method of observing cell cycle processes in a cytoplasmic extract in
vitro.
Key words cell-cycle ¡ amphibian ¡ oocyte ¡ zygote ¡
meiosis ¡ mitosis ¡ Maturation-Promoting Factor
(MPF) ¡ Cyto-Static Factor (CSF)
Introduction
It was half a century ago, when I was in my senior year
as a student of zoology, that animal development, cell
differentiation in particular, began to capture my imagination by its mysterious features and became the
most fascinating subject among those I was studying. I
learned from Hans Spemann’s book (Spemann, 1938; p.
Yoshio Masui
Department of Zoology, University of Toronto, 25 Harbord St.,
Toronto, Ontario M5S 3G5, Canada
e-mail: masui/zoo.utoronto.ca
Tel: π1 416 978 3493. Fax: π1 416 978 8532
U. S. Copyright Clearance Center Code Statement:
202) that development is the expression of gene activities
in embryonic cells. However, in those days, geneticists
were spending most of their time looking for genes that
control enzyme activities in metabolism. Cell biologists
were also busy describing fine structures and biochemical activities of the cell, being enchanted by powerful
techniques of cell analysis such as phase- and electronmicroscopy and cytochemistry. Embryologists were trying to understand the mechanism of development, but
still followed the 19th century tradition Entwicklungsmechanik (developmental mechanics) proposed by
Roux. One of their major problems was that of embryonic induction by the organizer in amphibian embryos,
which they were investigating using simple microsurgical
techniques. This approach to development seemed to
better suit my taste for science than genetic analysis of
metabolic activities and descriptions of fine structure
and chemistry of the cell. I chose the problem of embryonic induction for my graduate research project.
Then, one day, I came across the paper by Markert
published in 1958. In this article, he stated, that ‘‘the
preferred viewpoint in this presentation has pictured a
cell, when subjected to differentiating stimuli, as responding through the activation of a gene which then
initiates a new reaction’’ (Markert, 1958; p. 11). Reading
this article, I was impressed by his insight, namely differential gene activation in development, and was strongly
convinced that the essential process of development involved nucleocytoplasmic interactions in the cell. At that
time, most people in his lab were analyzing lactic acid
dehydrogenase (LDH) isoenzymes in various animals
and their changes during development. I asked Professor
Markert, who was busy as Chairman of the Department
of Biology at Yale University, to teach me how to approach the problem of cell differentiation through isoenzyme analysis. Thus, I joined his lab in 1966 on sabbatical leave.
He gave me packs of deep-frozen penguin embryos
0301–4681/2001/6901–1 $ 15.00/0
2
sent from the Antarctic by an expeditionary team and
suggested a project to clarify developmental changes in
penguin LDH isoenzyme pattern. After a half year of
hard work, I found that the penguin has 3 different
LDH subunits, generating 15 different tetramer isoenzymes, while mammals have 2 different subunits and 5
isoenzymes, but their developmental changes were too
complicated to deduce any rule of their differentiation
(Markert and Masui, 1969). This taught me that in order
to develop even a single enzyme, embryonic cells must
undergo complex changes in gene expression. At this
point, Markert said ‘‘That’s enough’’, and suggested that
I should work on a project in which I am really interested, but which would be inexpensive so that I could
continue in Japan.
The experience with penguin LDH led me to the
pessimistic view that it was impossible to study cell differentiation at the molecular level using the 1960s’ technology. I was not convinced that all biochemical
methods available at that time were sensitive enough to
analyse such subtle biological processes as embryonic induction. Many embryologists also seemed to have lost
confidence in discovering the mechanism of cell differentiation for other reasons. First, the discovery that a variety of chemically unrelated substances could act as the
inducer of embryonic induction made us suspicious
about the requirement for its specificity as the inducer
of cell differentiation. Secondly, being influenced by the
immunologists’ idea of clonal selection as the cause of
cell differentiation (Burnet, 1959), embryologists also
began to question the nature of the process of cell differentiation, whether it is cell transformation or clonal
selection by the inducer. To skirt around this question I
needed a cell system in which I could observe the differentiation behaviour of a single cell under the influence of
a well-defined inducer. I thought that oocyte maturation
could satisfy these requirements.
Oocyte maturation
It was in 1962 that I learned for the first time about
oocyte maturation, when Professor Nace of the University of Michigan visited my laboratory on his sabbatical
leave and showed me Heilbrunn’s classical experiment
(Heilbrunn et al., 1939). This experiment demonstrated
that a piece of frog ovary suspended in Ringer solution
could be induced to ovulate oocytes in vitro when a
macerated pituitary suspension was added to the solution. I also read Brachet’s comment on oocyte maturation in his book ‘‘Chemical Embryology’’ published in
1951: ‘‘This whole field, so interesting both from the
point of view of embryology and of cellular physiology,
remains to be explored’’ (p. 146). I surveyed the literature, reviewed previous work on oocyte maturation (Masui, 1967a), and learned the general features of oocyte
maturation in the animal kingdom.
In all animals, oocytes growing in the ovary are arrested in the prophase of the first meiosis (P I), the G2
phase of the cell cycle. Fully grown oocytes resume meiosis when oocyte maturation begins, leading to the formation of an egg which can be fertilised (Fig. 1). In
some species, such as the surf clam, Spisula solidissima
and the echiuroid, Urechis caupo, oocyte maturation is
initiated by fertilisation after the oocytes are spawned.
However, in most animals oocyte maturation is initiated
prior to ovulation when the ovary is stimulated by
gonadotropin secreted from the pituitary in vertebrates
or from nerves in invertebrates. Therefore, in the frog,
oocyte maturation is induced by the special signal, pituitary gonadotropin, and has a well-defined endpoint, egg
maturity. In addition, all changes during oocyte maturation can be observed in a single cell, the oocyte.
The first visible sign of oocyte maturation is ‘‘germinal vesicle breakdown (GVBD)’’, when the large nucleus
of the oocyte, called the germinal vesicle (GV), breaks
down to release its nucleoplasm into the cytoplasm. This
is followed by chromosome condensation to a metaphase state before the first meiotic division. If oocyte
maturation is initiated by fertilisation, as in some invertebrate species as mentioned above, the oocyte completes
two meiotic divisions without interruption, which is immediately followed by mitosis. If oocyte maturation is
induced by gonadotropin before fertilisation, three different cases are distinguished: mature oocytes arrest 1)
at metaphase of the first meiosis (M I) as in the insect
and tunicate, 2) at metaphase of the second meiosis (M
II) as in vertebrates, and 3) in G1 phase at the pronuclear stage as in sea urchins and jelly fish. In all these
cases, for the eggs to be relieved from the arrest, complete meiosis, and proceed to mitosis they must be activated by fertilisation or artificial stimuli (Fig. 1).
Markert often talked about the economic value of
cloning homozygous animals from diploid oocytes of a
good strain by suppressing meiotic divisions followed by
parthenogenesis. To this end, he suggested that I might
try to find a way to suppress meiosis in frog oocytes.
However, I thought that in order to suppress meiotic divisions, I should know how oocyte cell cycles are regulated. Thus, I realised that frog oocyte maturation would
be a highly advantageous system over other cell systems
for studying control of cell cycle events, because I could
use stimuli that induce oocyte maturation and egg activation as separate signals to drive the cell cycle from G2
to M and then from M to G1, respectively. This could
eliminate the problems of synchronizing cell cycles, always involved when we study the cell cycle in multicellular systems and therefore allow us to handle cells at a
specific cell cycle stage with high accuracy.
For my study, I chose oocytes of Rana pipiens, the
most common frog in North America and easily available from local dealers. The fully grown oocytes are easy
to culture in a simple salt solution such as Ringer’s, and
their large size ranging from 1.5 to 2.0 mm in diameter
3
Fig. 1 Progression of meiosis
during oocyte maturation and
egg activation by fertilisation
(Masui and Clarke, 1979). Oocytes of the animals indicated
are arrested and waiting for fertilisation at the stages of meiosis shown in the diagram:
GV – germinal vesicle;
GVBD – germinal vesicle
breakdown.
and resistance to injuries allow us to operate microsurgically as demonstrated by successful nuclear transplantation experiments (DiBerardino, 1997 for a review).
Therefore, I hoped to develop a subcellular Entwicklungsmechanik of nucleocytoplasmic interactions in an
inexpensive way, but also I thought that it would not be
difficult to perform biochemical analysis as well, because
a large quantity of oocytes at a same phase of the cell
cycle can easily be obtained.
Nucleocytoplasmic relations in oocyte maturation
The analysis of relations between the nucleus and cytoplasm in development has a long history. It was Hertwig
(1903 and 1908) who first brought to our attention the importance of nucleocytoplasmic relations for cell physiology. He interpreted alternation of cell division and cell
growth as a homeostatic mechanism that maintains nucleocytoplasmic ratio; but he also pointed out exceptions,
such as the oocyte which grows without division and the
zygote which divides without growth. There had been accumulated many classical observations on oocytes and
zygotes in various animals which showed essential roles of
nucleocytoplasmic relations in cell division and differentiation (Gurdon and Woodland, 1968, for a review).
It was at the beginning of the 20th century that embryologists discovered the important role of the nucleoplasm of the GV in cell divisions of the zygote as reviewed by Wilson (1925). They fertilised oocyte fragments
lacking the GV which had been separated from an oocyte before GVBD. These cytoplasmic fragments prevented from receiving nucleoplasm from the GV failed
to initiate mitosis. On the other hand, fragments con-
taining the GV or those separated after GVBD which
had received nucleoplasm could undergo mitosis and develop (Fig. 2). In the sea urchin and frog, it was also
found that when prematurely inseminated eggs failed to
be activated, egg chromosomes remain at metaphase and
sperm chromosomes incorporated into the egg cytoplasm are also condensed to a metaphase state (Gurdon
and Woodland, 1968, for a review). This observation
suggested that the cell cycle activity of the nucleus, either
of the egg or of the sperm, conforms to that of maturing
oocyte cytoplasm.
However, no further studies on this problem had been
carried out until the early 1960s. Even in 1967 when I
began my study of oocyte maturation, I could find no
more than a few papers which dealt with the nucleocytoplasmic interaction during maturing oocytes. In 1961,
Subtelny and Bradt showed that somatic nuclei transplanted into Rana pipiens oocytes at M II underwent
mitosis if the recipient oocytes were activated, whereas
those transplanted before M II did not. In 1964, Dettlaff
and her associates, using toad oocytes, demonstrated
that while oocytes maturing with the GV could develop
the cell surface contractility to become capable of cell
division, those whose GV had been removed beforehand
fail to do so. Thus, they confirmed the discovery made
60 years ago on the role of GV nucleoplasm in cell divisions in marine invertebrate oocytes as mentioned before. In 1966, Iwamatsu, using the medaka fish, found
that the GV failed to break down in gonadotropinstimulated oocytes if it had been displaced from the hyaline cytoplasm into yolky cytoplasm by a centrifugal
force. This suggested that the factor which could cause
GVBD was not the hormone itself but the hyaline cytoplasm stimulated by the hormone.
4
Fig. 2 Experimental scheme
for testing the role of nucleoplasmic substances (dots) derived from the germinal vesicle
(GV) in promoting cell division
of fertilised eggs of marine invertebrates (Cerebratulus and
starfish) (Wilson, 1925).
In the latter half of the 1960s, more studies on cytoplasmic control of nuclear activities appeared (Gurdon
and Woodland, 1968; De Terra, 1969; Johnson and Rao,
1971 for reviews). Gurdon, Graham, and their associates
using Xenopus laevis demonstrated that somatic nuclei
transplanted into a zygote are induced to synthesize
DNA and enter mitosis synchronously with the host cell;
those put into ovarian oocytes cease DNA synthesis and
initiate RNA synthesis, and those put into maturing oocytes to condense chromosomes to a metaphase state
(Gurdon, 1968). Clearly, activities of transplanted nuclei
always conform to those of the host cells regardless of
their original activities in the donor cells, indicating the
strong control of the cytoplasm over the nucleus. Similar
evidence for the cytoplasmic control over the cell cycle
activity of the nucleus had also been provided by exchanging the nucleus or cytoplasmic fragments in protozoa (De Terra, 1969) or by fusing cultured mammalian
cells (Johnson and Rao, 1971) in different cell cycle
phases. However, all these studies provided us with no
clue to the nature of the factor involved in the cytoplasmic control – whether it is the chemical activity of
a specific molecule in the cytoplasm or a physiological
function of the cytoplasm as a whole.
Hormonal regulation of oocyte maturation
Although the oocytes of marine invertebrates, which
mature and can be fertilised in sea water, had been excellent materials for embryological as well as cytological
studies since the late 19th century, the natural cause of
oocyte maturation in these animals was unknown for
a long time, except for cases of maturation induced by
fertilisation. On the other hand, in 1935, Pincus and
Enzman found in mammals that fully grown oocytes
isolated from the ovarian follicle could mature spontaneously when cultured in vitro. This suggested that removal of the oocyte from the maturation-inhibiting effect of the follicular environment by ovulation is the
cause of oocyte maturation. However, in 1939, Heilbrunn et al., using the frog as mentioned before, demonstrated for the first time that ovulation and oocyte maturation could be induced by pituitary gonadotropin.
Later, it was also found that the effect of gonadotropin
could be augmented by progesterone (Zwarenstein,
1937; Burgers and Li, 1960; Wright, 1961). It was not
until the late 1950s that gonadotropin was discovered in
the invertebrates, when gonad-stimulating substance was
extracted from radial nerves of the starfish and shown
to induce ovulation and oocyte maturation (Chaet and
McConnaughty, 1959; Kanatani, 1964).
However, even in the early 1960s it was unclear how
these hormones act on the oocyte. Therefore, the study
published in 1964 by Dettlaff and her colleagues bore
the most important significance to answering this question. They isolated toad oocytes from the ovary by removing the surrounding follicular epithelium with forceps and cultured them in Ringer solution containing a
suspension of macerated pituitary. These oocytes were
found to acquire ‘‘maturation inertia’’ just before
GVBD and produce the nucleoplasmic substance in the
5
Fig. 3 Experiments showing relative roles of
pituitary gonadotropin, follicle cells, and progesterone in the induction of maturation of
frog oocytes (Masui, 1967b).
GV that the authors showed to have the ability to induce
maturation when injected into other oocytes without
hormonal stimulation. Thus, once oocytes acquire
maturation inertia they no longer require the presence of
the hormone in the medium to continue the subsequent
maturation process. From these results it was concluded
that the hormone directly acts on the oocyte nucleus to
induce new gene activities. This conclusion seemed to
conform to the view of hormonal actions on the cell
widely popular in those days, that is, gene activation by
hormones (Davidson, 1965).
In early 1967, I repeated these experiments using
Rana pipiens. I observed that when intact ovarian fragments were treated with gonadotropin, fully grown oocytes undergoing GVBD were shed from follicles (Fig.
3a). However, when I isolated oocytes manually from
the ovary by removing follicular epithelium with forceps,
I found under a dissection microscope at high magnification that many cells still adhered to the oocyte surface.
It was these oocytes that remained responsive to the hormone (Fig. 3b). To obtain oocytes completely free of
follicle cells, oocytes had to be washed in Ca-Mg-free
Ringer solution to which EDTA was added. These clean
oocytes were found to be no longer responsive to the
hormone (Fig. 3c). However, they were capable of maturation when incubated with follicle cells in the presence
of pituitary extract (Fig. 3d). Therefore, I concluded
that the effect of gonadotropin must be mediated by follicle cells to induce frog oocytes to initiate maturation.
Since progesterone had been known to have the effect
on the ovary that facilitates ovulation in the frog as mentioned before (Zwarenstein, 1937; Burgers and Li, 1960;
Wright, 1961), I tested the effect of progesterone on oocytes free of follicle cells as well as on those enclosed in
the follicular epithelium, either in the presence or in the
absence of gonadotropin. The results showed that progesterone could induce oocytes to mature in all these
cases (Fig. 3e). These mature oocytes proved to be capable of cleavage when a blastula nucleus was transplanted into them. Therefore, I concluded that ‘‘pituitary gonadotropin acts on the follicle cells to stimulate
them to release a hormone that directly acts on the oocyte’’ (Masui, 1967b; p. 365), and that ‘‘the follicle cells
secrete a progesterone-like substance that causes maturation of oocytes (Masui, 1967b; p. 374).
Progesterone action on frog oocyte maturation was
6
also reported independently by Schuetz (1967) and
Smith et al. (1968). In 1967, the production of a substance in the starfish ovary stimulated by neural gonadotropin that causes oocyte maturation was also reported
by Kanatani and Shirai and by Schuetz and Biggers. In
1969, this substance was identified as 1-methyl-adenine
(1-MA) by Kanatani et al. However, in the frog, it was
1975 when radio-immunoassay became commonly available that progesterone was identified as the natural inducer of oocyte maturation by Fortune et al. (1975).
To know how progesterone acts upon the oocyte, I
injected a progesterone solution into Rana pipiens oocytes to give an internal concentration of 0.5 mg/ml, but
none of the oocytes were induced to mature (Fig. 3f),
while those treated by applying externally a less concentrated progesterone solution all matured (Masui and
Markert, 1971). Similar observations were reported by
Smith and Ecker (1969) in frog oocytes treated with progesterone and by Kanatani and Hiramoto (1970) in starfish oocytes treated with 1-MA. All these observations
indicated that the hormone becomes effective in inducing oocyte maturation only when its action is exerted
from outside of the oocyte or on the oocyte surface. Although this conclusion was contrary to the above-mentioned notion of hormone action at that time, it gained
strong support several years later from the experiments
with polymer-conjugated progesterone derivatives (Ishikawa et al., 1977; Godeau et al., 1978). These experiments in Xenopus showed that the steroid applied externally could induce oocyte maturation, even though prevented from entering the oocyte because of the
conjugated polymer, but it failed to induce oocyte maturation if injected into oocytes.
However, the action of progesterone as maturation inducer in amphibian oocytes did not seem to be as highly
specific as I initially expected. In fact, various steroid
hormones and non-steroid substances were found to be
capable of inducing oocyte maturation at varying efficiency (Masui and Clarke, 1979 for a review). Interestingly, all these agents were found to be effective only
when they were applied to the oocyte from the outside.
When various chemical agents which could specifically
affect cell physiology, such as ion channel blockers,
ionophores, and chelators, became available between
1975 and 1980, these substances were tested for the ability to induce or inhibit oocyte maturation in various species. Treatment of oocytes with Ca ionophore A23187 in
the presence of Ca and Mg ions at a high concentration
could induce oocyte maturation in various animals.
Many Ca-releasing agents were known as maturation inducers, including ionophoresis of Ca ions, b-adrenergic
blockers, and lanthanum ions. In Xenopus, insulin and
insulin-like growth factor were later found to induce oocyte maturation (El Etr et al., 1980; Maller and Koontz,
1981).
Conversely, it was shown that oocyte maturation
could reversibly be inhibited by removal of Ca ions in
external medium or by treatment with Ca-channel
blockers as well as with Ca ion chelators such as EGTA
(Masui and Clarke, 1979 for a review). When Ca metabolism in oocytes was examined using radioactive Ca
and Ca-activated chemiluminescent protein, aequorin,
Ca ion release from oocytes was observed at the beginning of oocyte maturation in Xenopus, Urechis caupo,
and starfish (Masui and Clarke, 1979 for a review).
These results strongly suggested the involvement of Ca
ions in the initiation of oocyte maturation. However,
later experiments with new techniques detected no significant changes in Ca ion levels in the beginning of oocyte maturation in starfish (Eisen and Reynolds, 1984)
and Xenopus (Robinson, 1985). Since then, the role of
Ca ions in the initiation of oocyte maturation has no
longer attracted serious attention. Nevertheless, our recent re-investigation of the Ca problem confirmed some
of the old observations mentioned above (Duesbury and
Masui, 1996). Thus, it may be suggested that there
should be further investigation into the role of Ca ions
in oocyte maturation.
Discovery of Maturation-Promoting Factor (MPF)
As mentioned before, progesterone could induce oocyte
maturation only when applied externally, but it failed to
do so if injected into the oocyte. This result suggested
that only the cytoplasm near the oocyte surface could
receive the hormonal signal. Therefore, it was assumed
that the oocyte must then create a signal in the cytoplasm that reaches the GV to induce meiotic divisions
(Masui and Markert, 1971). This implies that nuclear
activities involved with the initiation of oocyte maturation are under the control of cytoplasmic activity, but
not vice versa. To test this assumption, I sucked cytoplasm from progesterone-treated oocytes into a calibrated graduated micropipette with the aid of a micromanipulator and injected a measured volume of the
cytoplasm into an untreated immature oocyte. The recipient oocytes were indeed induced to mature without
hormone treatment when they were injected with more
than 5 nl of cytoplasm taken from the donor oocytes
which had been kept for more than 6 h at 21 æC after
hormone treatment (Fig. 4A). This indicated that the
frog oocyte, having received the progesterone signal near
the cell surface, produced a factor in the cytoplasm that
causes oocyte maturation, and this cytoplasmic factor
was named ‘‘Maturation Promoting Factor (MPF)’’.
These results were published in 1971. In the same year,
Smith and Ecker also reported a similar result in the
discussion of their paper that investigated the action of
progesterone. They suggested, based on their preliminary experiment, that ‘‘maturing oocytes acquire an intracellular component which in itself is capable of inducing maturation’’ (Smith and Ecker, 1971; p. 245).
I found that if the cytoplasm taken from progester-
7
Fig. 4 Transfer of cytoplasm from mature
oocytes with or without the GV into immature oocytes or into a blastomere of a 2-cell
embryo to demonstrate the nucleus-independent production of MPF and CSF
(Masui and Markert, 1971).
one-treated oocytes at a same stage of maturation was
injected into untreated oocytes, the frequency with
which recipient oocytes were induced to mature increased almost linearly with the volume of the injected
cytoplasm. Based on this dose-dependent relationship,
the relative activity of MPF in oocyte cytoplasm, being
expressed as the percentage of oocytes induced to mature by injection with a constant volume of cytoplasm,
was measured at various times after the initiation of
maturation by progesterone treatment. Thus, it was
found that MPF appeared 6 hours after progesterone
treatment, 3 hours before GVBD, stayed at high levels,
and then declined after egg activation by fertilisation or
pricking with a glass needle (Fig. 5). However, low MPF
activity could still be detected in cleaving blastomeres of
zygotes (Masui and Markert, 1971).
Since the surface action of progesterone caused the
appearance of MPF and surface injuries of activated
eggs caused its disappearance, it was supposed that both
appearance as well as disappearance of MPF must be
initiated by cytoplasmic events near the cell surface
without involvement of the nucleus. This was proved by
experiments with oocytes whose GV had been removed
prior to progesterone treatment (Fig. 4B). When treated
with progesterone, the cytoplasm of these enucleated oocytes produced MPF, but when pricked with a glass
needle, the oocytes were activated and their cytoplasm
lost MPF activity (Masui and Markert, 1971).
On the basis of these results, it was supposed that
MPF must reach the nucleus from the oocyte surface by
propagating itself in the oocyte cytoplasm by its ‘‘autocatalytic’’ amplification. This was corroborated by a
serial transfer of cytoplasm in Rana pipiens oocytes. In
this experiment, 30 to 40 nl (1.5 to 2.0 % of oocyte volume) of cytoplasm were transferred from progesteronetreated donor oocytes to untreated recipient oocytes,
and then from these first recipients to the second recipients and from the second to the third, successively at
one day intervals (Fig. 6). The cytoplasm thus transferred was able to induce maturation in the recipients
after every transfer with similar frequency (70 to 90 %),
showing no decrease in MPF activity in the serially
transferred cytoplasm, despite the fact that the cytoplasm of the original progesterone-treated donors was
extensively diluted through the serial transfers (Masui
and Markert, 1971).
I also examined actual propagation of MPF activity
through oocyte cytoplasm from the animal to the vegetal
half of the oocyte. It was found that in progesteronetreated oocytes MPF developed earlier and its activity
increased faster and became higher in the animal half
than in the vegetal half. If the GV in an oocyte was
displaced by centrifugation to the vegetal half and the
oocyte was then constricted by thin thread along the
equatorial line and treated with progesterone, there was
a significant delay in GVBD and the frequency with
which such oocytes underwent GVBD was decreased in
proportion to the tightness of constriction (Masui,
1972).
A few years later, the presence of MPF and its autoca-
8
Fig. 5 The time-course of
MPF and CSF activities during
oocyte maturation, egg activation, and early cleavage
stages.
Fig. 6 A serial transfer of cytoplasm from a maturing oocyte
into an immature oocyte demonstrating ‘‘autocatalytic’’
amplification of MPF (Masui
and Markert, 1971).
talytic amplification were reported in Xenopus oocytes,
but it was not until 1976 that MPF was found in oocytes
of a variety of non-amphibian species, including the
starfish, mouse and Spisula (Masui and Clarke, 1979, for
a review).
Protein synthesis and phosphorylation during
oocyte maturation
In 1966 Smith et al. observed that oocytes significantly
increase protein synthesis during maturation, and
Dettlaff (1966) showed that gonadotropin-induced
maturation of follicle-enclosed oocytes is inhibited by
both RNA and protein synthesis inhibitors. However,
in 1967 Schuetz found that progesterone-induced oocyte
maturation was not inhibited by RNA synthesis inhibitors but only by protein synthesis inhibitors, suggesting
that oocytes require only translation of stored mRNA
to initiate maturation.
In 1975, William Wasserman, who was working towards his PhD in my lab, defined a biological unit of
time for progression of oocyte maturation, referring to
the duration between the beginning of progesterone
9
treatment and the time when 50 % of the oocytes initiate
GVBD as GVBD50. Using Xenopus oocytes he showed
that GVBD could be inhibited only in the oocytes injected with protein synthesis inhibitors no later than 65 %
of the GVBD50, which coincided with the time of the
initial appearance of MPF in 50 % of the oocytes. It was
also found that protein synthesis inhibition could not
stop the oocyte maturation induced by MPF injection,
unlike that induced by progesterone. Furthermore, protein synthesis-inhibited oocytes when injected with cytoplasm containing MPF could produce as much MPF as
that produced in uninhibited oocytes. These results indicated that once MPF appears in the cytoplasm both the
amplification process as well as the action of MPF to
promote oocyte maturation are no longer dependent on
protein synthesis. Therefore, it was hypothesized that the
initiation of oocyte maturation by progesterone requires
synthesis of a new protein, ‘‘initiator’’, and this leads to
activation of the MPF precursor protein stored in the
oocyte, so-called pre-MPF. Therefore, once active MPF
appears, it is autocatalytically amplified by further activation of pre-MPF with no protein synthesis required to
continue the subsequent process of maturation (Wasserman and Masui, 1975).
In 1975, Drury and Schorderet-Slatkine also independently reported an experiment similar to ours. However,
their serial cytoplasmic transfer experiment showed progressive decreases in MPF activity in the cytoplasm successively transferred from donor to recipient oocytes
when the oocytes were treated with protein synthesis inhibitor. Therefore, they suggested protein synthesis dependency of MPF amplification. However, our results
with Xenopus oocytes were later confirmed by other laboratories (Detlaff et al., 1977; Gerhart et al., 1984). In
1988, Sagata and his associates in Vande Woude’s lab
identified ‘‘initiator’’ protein as Mos, the product of the
proto-oncogene c-mos, and showed that this is the only
protein to be synthesised in Xenopus oocytes to produce
MPF.
In the mid 1970s, it became known in many animals,
including mammals and starfish, that the induction of
GVBD does not require protein synthesis at all, not even
an ‘‘initiator’’ (Masui and Clarke, 1979, for a review).
Clearly, in these animals, it is not the synthesis of a new
protein, but a modification of a pre-existing protein that
is required for the appearance of active MPF. Then, a
rapid increase in protein phosphorylation was observed
in the initial phase of maturation of starfish (Guerrier
et al., 1977) and Xenopus oocytes (Maller et al., 1977).
In the frog, this protein phosphorylation surge could be
induced by injection of MPF as well as by hormonal
stimulation (Maller et al., 1977), but it was preceded by
a rapid decrease in cAMP levels (Speaker and Butcher,
1977). In addition, it was found that GVBD could be
induced by injection of a specific inhibitor of cAMPdependent protein kinase (PKA) (Maller and Krebs,
1977). From these observations, it was inferred that it is
a switch of protein phosphorylation from that catalysed
by PKA to the one catalysed by other protein kinases
that is required for activation and amplification of MPF
(Masui and Clarke, 1979, for a review).
However, in the late 1970s, the existence of pre-MPF
was questioned in Rana pipiens (Schuetz and Samson,
1979) and Acipenser stellata, the sturgeon (Detlaff et al.,
1977). In these animals, the appearance as well as amplification of MPF do require protein synthesis. In fact,
these classical observations have been corroborated by
recent molecular studies by Yamashita and his associates
in a variety of fish and amphibian species. They found
that oocytes of most species do not have pre-MPF, and
must synthesize MPF de novo following hormonal
stimulation to initiate maturation (Yamashita, 1998, for
a review).
Discovery of Cytostatic Factor (CSF)
Having detected low MPF activities in cleaving blastomeres as mentioned before, I became suspicious about
the specificity of MPF as promoter of oocyte meiosis
and curious about a possible role in mitosis. To explore
this possibility, cytoplasm from oocytes which had completed GVBD was injected into blastomeres of 2-cell embryos. To my surprise, ‘‘the injected blastomere frequently stopped cleaving. This result was unexpected’’
(Masui and Markert, 1971; p. 135) (Fig. 4A). On the
other hand, cytoplasm taken from oocytes before
GVBD or fertilised eggs did not inhibit cleavage when
it was injected into blastomeres of 2-cell embryos. This
indicated that only the oocytes that have passed M I and
of those arrested at M II possess an inhibitory factor in
their cytoplasm that stops cleavage of blastomeres.
Thus, I hypothesized that maturing oocytes produce in
the cytoplasm a factor responsible for the arrest of oocyte meiosis at M II, and its removal by fertilization
allows zygotes to cleave. This hypothetical factor I called
‘‘Cyto-Static Factor (CSF)’’ in 1971 (Masui and
Markert, 1971).
Relative activities of CSF in oocyte cytoplasm could
also be assayed in the same way as MPF based on the
proportionality between the volume of the cytoplasm injected into a blastomere and the frequency with which
blastomeres were arrested with the injected cytoplasm.
Thus, it was found that CSF activity appears after M I,
gradually increases, and remains high as oocytes mature,
but abruptly decreases shortly after fertilization. However, unlike MPF, it never appears again in cleaving zygotes (Fig. 5). These changes of CSF, both its production
during oocyte maturation as well as its destruction during egg activation, were found to occur independently
of nuclear activities, since CSF appeared in enucleated
oocytes when they were treated with progesterone (Fig.
4B) and disappeared when they were activated by pricking with a glass needle (Masui and Markert, 1971).
10
In 1980, we proposed the criteria for CSF to be the
genuine inhibitor of the cell cycle (Masui et al., 1980;
Masui, 1991; 2000 for reviews). First, as shown above,
CSF should be found only in oocytes arrested at M II.
Secondly, the zygotes arrested by CSF should have the
same cytological as well as physiological properties as
those of the unfertilised egg. Indeed, both CSF-arrested
blastomeres and unfertilised eggs were found to contain
a mitotic apparatus equipped with a well-formed spindle
and condensed chromosomes aligned at the equator, but
associated with no polar asters, showing characteristics
of meiotic metaphase. As well, the cytoplasm of both
CSF-arrested zygotes and unfertilised eggs were found
to have MPF and chromosome-condensation activity
that causes brain or sperm nuclei injected into these cells
to condense chromosomes to a metaphase state. Also,
the surface morphology of CSF-arrested blastomeres becomes similar to that of unfertilized eggs, forming
microvilli which are absent in blastomeres undergoing
mitosis (Masui et al., 1980).
Next, CSF must be inactivated during the process of
egg activation. In Rana pipiens, this process occurs in the
first 45 min following fertilisation (Meyerhof and Masui,
1977). Zygotes injected with unfertilized egg cytoplasm
during this period continued to cleave, whereas those
injected 60 min after fertilisation, or later, stopped cleaving. It appears that injected CSF is inactivated during
the early period of egg activation together with the endogenous CSF of the recipient zygote. As seen in the
following section, CSF can also be destroyed in vitro
under the physiological condition that brings about egg
activation.
Finally, inhibition of the cell cycle by CSF should be
reversible: CSF-arrested blastomeres must resume cell
cycles when artificially activated as unfertilized eggs do.
Indeed, CSF-arrested blastomeres initiated DNA synthesis and mitosis as well as exhibited oscillation of
Fig. 7 Stratified oocyte of Rana pipiens. Progesterone-treated oocytes were centrifuged in a Ficoll solution at 4080 ¿ g for 2 h (Masui, 1972).
MPF activity in the cytoplasm when the blastomeres
were injected with a large number of sperm nuclei or Ca
ions or treated with ionophore A23187. These observations ruled out the suspicion that CSF is a non-specific toxic substance (Masui, 1991; 2000 for reviews).
Extraction and chemical characterization of MPF
and CSF
MPF was found to remain active for a short while in
cytoplasmic homogenates prepared from mature oocytes
after brief homogenization with a glass needle in a polyethylene tube (Masui and Markert, 1971). However,
when the homogenates were centrifuged, neither MPF
nor CSF activity was detected in the supernatant even
under the conditions that minimise protein denaturation. After two years of unsuccessful trials to extract
these cytoplasmic factors, I suspected that they might be
structural elements of cytoplasm or a substance firmly
bound to them. In 1972, I carried out an experiment to
determine the subcellular localization of MPF. In this
experiment, mature oocytes floated over a Ficoll solution were gently centrifuged, and each layer of cytoplasm
stratified in an unbroken oocyte (Fig. 7) was tested for
MPF activity by injecting its content into immature oocytes. The strongest MPF and CSF activities were detected in the cytosol and hyaline cytoplasm (Masui,
1972; 1974).
I then realised that ‘‘homogenisation’’ followed by
centrifugation was detrimental to MPF and CSF activities. This was quite understandable in view of the fact
that mature eggs activated by even a slight injury such
as pricking with a glass needle lose both MPF and CSF
activities. Therefore, dejellied eggs packed in a tube were
compressed by centrifugal force without homogenisation
and cytosols were extracted. In this way, in 1974, I succeeded in extracting CSF without losing its activity in
cytosols (Masui, 1974). However, the activity was still
short-lived in the cytosols kept in a refrigerator.
In order to protect MPF and CSF further from the
effect of egg activation, which might have occurred during the process of extraction, cytosols were extracted in
a Ca-free buffer containing a Ca-chelator, EGTA, since
a release of intracellular Ca2 π was known to be a universal cause of egg activation (Jaffe, 1985, for a review).
The cytosols extracted in this way were found to retain
high MPF and CSF activities for at least three days in
a refrigerator. This allowed us to perform chemical tests
on these factors. Bill Wasserman, working for his PhD
research in my lab, showed that MPF activity in cytosols
extracted from Rana pipiens oocytes was quickly lost
when EDTA, a Ca-Mg chelator, as well as Ca were
added and that it could be inactivated by proteolytic enzymes, but not by RNAses, and sedimented into 3 different fractions with sedimentation constants 3S, 13S, and
30S. From these observations, he concluded that MPF
11
is a protein existing as, or associated with, molecules of
different sizes and that its activity is sensitive to Ca and
dependent on Mg ions. These results were published in
1976 (Wasserman and Masui, 1976). In 1978, Drury succeeded in further stabilising MPF by adding ATP and
protein phosphatase inhibitors to Xenopus egg extracts.
Thus, in 1979, Masui and Clarke speculated that MPF
must be a phosphoprotein, and ‘‘MPF activity is maintained by phosphorylation of its molecules’’ (Masui and
Clarke, 1979; p. 246). In 1980, Wu and Gerhart succeeded in partial purification of Xenopus MPF using
ammonium sulphate precipitation and column chromatography and found that the MPF samples purified 30to 50-fold, containing 70 to 250 kD polypeptides, became less Ca-sensitive and were constantly phosphorylated and dephosphorylated in the presence of ATP.
Peter Meyerhof, carrying out his PhD research in my
lab, found that addition of Ca2π to or removal of Mg2π
from the cytosols quickly causes the loss of CSF activity.
These Ca-containing cytosols in a vial kept on ice lost
their activity completely within one hour of Ca addition
to the level of 10ª5 M. From these results, we hypothesised that the natural cause of CSF inactivation during
egg activation is the release of Ca ions from cytoplasm.
However, in the process of these studies, we noticed the
curious fact that CSF activity reappeared in Ca-containing cytosols during storage in a refrigerator. Thus, we
discovered to our surprise that there were two kinds of
CSF, the Ca-sensitive one and a Ca-insensitive one which
could only develop in stored egg cytosols after the former was inactivated by addition of Ca to the cytosols.
To distinguish these two CSFs, we labelled the former
‘‘primary CSF (1 æCSF)’’ and the latter ‘‘secondary CSF
(2 æCSF)’’. Their effects were cytologically indistinguishable; both bringing about metaphase arrest of the same
feature as described before. However, we observed an
essential difference between them: while 1 æCSF injected
into eggs shortly after fertilization failed to arrest cleavage of the recipient eggs as I mentioned before, Ca-resistant 2 æCSF could arrest cleavage regardless of the time
of its injection. This strengthened our hypothesis proposed above that Ca ion released from the egg early in
fertilisation is responsible for inactivation of Ca-sensitive 1 æCSF. We published these results in 1977 (Meyerhof and Masui, 1977).
In 1988, Ellen Shibuya, working for her PhD research
in my lab, succeeded in further stabilizing 1 æCSF by
adding a phosphatase inhibitor, NaF, and ATP to egg
cytosols, but she also found that these chemicals could
enhance its activity (Shibuya and Masui, 1988). This
suggested that the activity of 1 æCSF is dependent on
protein phosphorylation. 1 æCSF was precipitated from
Rana pipiens egg cytosols by ammonium sulphate and
sedimented in fresh cytosols by centrifugation at 150,000
¿ g for 6 h (Shibuya and Masui, 1988) and through a
sucrose density gradient with a sedimentation coefficient
of 3S in the presence of ATP and NaF (Shibuya and
Masui, 1989). 1 æCSF was partially purified eight fold
through these procedures. When fresh egg cytosols were
incubated with proteases, the activity of 1 æCSF was lost,
but when incubated with RNAses it was not (Shibuya
and Masui, 1989). Therefore, we concluded that 1 æCSF
is a labile phosphoprotein and, like MPF, activated by
its Mg2 π-dependent phosphorylation but inactivated by
Ca2 π ions.
On the other hand, Ca-resistant 2 æCSF was found to
be a very stable molecule, which could be precipitated
with ammonium sulphate without loss of its activity in
the absence of ATP, and could not be inactivated even
in 8 M urea, 4 M LiCl, and alkaline solution (pH 11.5)
(Shibuya and Masui, 1989). 2 æCSF purified 40-fold by
gel filtration equilibrated with 8 M urea was found to
be a very large molecule, perhaps larger than 2,000 kD.
It was found that 2 æCSF could be digested by proteolytic enzymes but not by RNAses (Shibuya and Masui,
1989). Our recent study showed that egg cytosols failed
to form 2 æCSF when transglutaminase inhibitors or
anti-transglutaminase antibodies were added to the cytosols (Zhang and Masui, 1997). In all probability, 2 æCSF
is an artefactual product of protein cross-linking reaction catalysed by Ca-activated transglutaminase that
could occur in egg cytosols during storage.
The ubiquitous role of MPF in the control
of nuclear activity
In the late 1960s, Gurdon (1968) using Xenopus oocytes
showed that somatic cell nuclei injected into maturing
oocytes were transformed into metaphase chromosomes.
In 1973, this was confirmed in Rana pipiens oocytes by
David Ziegler, who was carrying out his graduate research in my lab. He found that oocytes acquire the ability to transform the nuclei transplanted into them to
metaphase chromosomes as MPF activity appeared in
their cytoplasm (Ziegler and Masui, 1973). In these oocytes, brain and sperm nuclei in the G1 phase were directly transformed to metaphase chromosomes consisting of a single chromatid (Fig. 8) (Ziegler and Masui,
1976a). These results strongly suggested that oocyte
cytoplasmic effects on nuclear activities are not tissuespecific. Neither did MPF seem to be a meiosis-specific
factor, since MPF activities were found to persist in
blastomeres at the cleavage stage as mentioned before
(Masui and Markert, 1971). We were discussing the
possible role of MPF in mitotic chromosome condensation. In 1978, Bill Wasserman, who left my lab to join
Smith’s lab at Purdue University as a postdoc in 1976,
demonstrated for the first time the periodic appearance
of MPF during mitotic cycles using blastomeres of amphibian embryos (Wasserman and Smith, 1978). His
finding was later re-examined and confirmed by Gerhart
et al. (1984).
By the end of the 1970s, it had also been established
12
Fig. 8 Chromosomes condensed to a metaphase state from brain
nuclei injected into a maturing oocyte with MPF (Ziegler and Masui, 1976a).
Fig. 9 Arrested blastomeres of three Xenopus laevis 2-cell embryos
injected with unfertilised egg cytoplasm of Rana pipiens (left) and
arrested blastomeres of three Rana pipiens 2-cell embryos injected
with unfertilised Xenopus laevis eggs (right) demonstrating the species-nonspecificity of CSF of amphibian egg cytoplasm (Meyerhof
and Masui, 1979).
that MPF from the oocyte of one species could induce
oocyte maturation in the other species, demonstrating
its species-nonspecificity (Kishimoto et al., 1982). Similarly, CSF from one amphibian species was found to arrest cleavage of blastomeres of another species (Meyerhof and Masui, 1979) (Fig. 9). Therefore, in 1979, we
concluded that ‘‘the nuclear events in oocytes undergoing meiotic divisions, and those in somatic cells undergoing mitotic divisions, are under the control of the
same cytoplasmic factors’’ (Masui and Clarke, 1979; p.
270). In the same year, the appearance of MPF during
mitosis was reported in HeLa cells by Sunkara et al.
(1979) and within a few years, in CHO cells by Nelkin
et al. (1980), in sea urchin embryos by Schatt et al.
(1983), and in yeast cells by Weintraub et al. (1982).
These results proved that MPF is a ubiquitous cytoplasmic factor that causes the cell division in eukaryotes.
In the late 1970s, by labelling proteins with radioactive amino acids, we investigated the interactions of the
nuclei transplanted into frog oocytes with proteins accumulated in the GV of host oocytes as well as with
proteins synthesized during their maturation. It was
found that these labelled proteins preferentially accumulated over the chromosomes that had been induced to
condense in maturing oocytes. On the other hand,
chromosomes condensed in maturing oocytes lacking
the GV disintegrated in less than 3 h. From these results,
we concluded that proteins synthesized in oocyte cytoplasm during maturation interact with the nucleus to
induce chromosome condensation, but proteins of GV
nucleoplasm are required for stabilisation of the condensed chromosomes (Ziegler and Masui, 1976b; Masui
et al., 1979).
The dose-dependent effect of M phase cytoplasm on
chromosome condensation of interphase nuclei was first
observed in a cell fusion experiment by Johnson and Rao
(1971) using cultured mammalian cells. In their experiments, chromosome condensation was induced with increasing frequency as the ratio of M phase cells fused to
interphase cells was increased. However, it remained unclear whether this dose-dependent effect of M phase cytoplasm was brought about by changes in its concentration
after mixing with the interphase cytoplasm or by changes
in the ratio between M phase cytoplasm and nuclei.
Therefore, we began our investigation into the effect of
nucleocytoplasmic ratio on chromosome condensation
induced by M phase cytoplasm in the early 1980s.
In 1982, Ellen Shibuya in her M.Sc. research observed
that sperm nuclei injected into a single CSF-arrested
blastomere of 2-cell frog embryos could be transformed
into metaphase chromosomes if their number per blastomere was kept below 100, while they formed pronuclei
and began DNA synthesis if the number exceeded 200
(Shibuya and Masui, 1982). Later, Clarke in his PhD
research investigated the behaviour of sperm nuclei in
polyspermic mouse oocytes arrested at M II following
premature insemination. He found that sperm nuclei
could be transformed into metaphase chromosomes if
the egg received no more than 3 sperm, but if the egg
received more than 3 sperm, they all form a pronucleus;
this nucleocytoplasmic ratio (3 nuclei/one oocyte cytoplasm) critical for sperm chromosome condensation remained unchanged if oocytes were bisected or fused.
From these results, we concluded that ‘‘the oocyte cytoplasmic factor which transforms the sperm nucleus into
metaphase chromosomes may be stoichiometrically titrated by sperm nuclear material’’ (Clarke and Masui,
1987; p. 838).
Cell cycles in vitro
In the late 1970s, I thought that in order to study the
nucleocytoplasmic interactions involved in the cell cycle
13
at the molecular level, we must investigate the behaviour
of nuclei in cytoplasmic environments better chemically
defined than the cytoplasm of intact oocytes and eggs.
Previously, such studies were attempted by incubating
Xenopus erythrocyte nuclei (Barry and Merriam, 1972)
and sea urchin sperm nuclei (Kunkel et al., 1978; Eng
and Metz, 1980) with egg homogenates or cytosols of
the respective species in vitro. However, they did not succeed in inducing significant changes in nuclear morphology associated with the cell cycle.
In 1978, I was trying to stabilize MPF in cytosols extracted from Rana pipiens eggs. So far, cytosols prepared
by high speed centrifugation (150,000 ¿ g for 2 h) always
had lost MPF activity on day 3 of cold storage on ice.
However, when I accidentally assayed MPF activity in the
cytosols on day 4, I found, to my surprise, that there was
still activity. When I continued to assay MPF activity
every day, the activity was found until it disappeared on
day 8, but then curiously it reappeared on day 9 and disappeared on day 13 and so on. That is, MPF activity actually
oscillated with fairly regular periods of 4 to 6 days. Oocyte
maturation induced by these oscillating MPF activities
appeared to be genuine in all respects (Fig. 10). I repeated
the experiment and confirmed that MPF activity in egg
extracts oscillated with the average period of 5.05 ∫ 1.25
day during storage at 2 æC. In 1982, I published these results and concluded that ‘‘a homogeneous cell-free system
can exhibit cyclic behaviour of a cytoplasmic factor controlling nuclear activities’’, and suggested ‘‘the possibility
that a modification of the system may allow MPF to oscillate at a higher temperature with a periodicity similar to
that of zygotes’’ (Masui, 1982; p. 397).
However, when I incubated frog brain nuclei in clear
egg cytosols obtained by high speed centrifugation in
which MPF activity cycled, although a high MPF activity was found in the cytosols, no chromosome condensation was induced. On the other hand, Manfred Lohka,
who chose this project for his PhD research, prepared
egg extracts which contained cytoplasmic granules by
low speed centrifugation (9,000 ¿ g, for 15 – 30 min) and
incubated demembranated sperm nuclei in the extracts.
Sperm nuclei seemed to be a better choice than brain
nuclei, since in nature only the sperm nuclei have been
designed to interact with egg cytoplasm. Thus, Lohka
succeeded in making pronuclei from sperm nuclei in vitro in the extract prepared from eggs which had been
activated by electric shock. These pronuclei replicated
DNA and subsequently formed metaphase chromosomes in the extract. Thus, he demonstrated, for the first
time, that the cell cycle events could take place in vitro.
In 1983, we published these results and concluded that
‘‘this cell-free system may be useful in biochemical
analysis of the interactions of nucleus and cytoplasm
that control nuclear behavior’’ (Lohka and Masui, 1983;
p. 719). A year later, a similar cell-free experiment using
toad eggs and sperm nuclei was also reported by Iwao
and Katagiri (1984).
Fig. 10 Rana pipiens oocytes induced to mature by injection with
a cold-stored egg extract in which MPF activity was oscillating
(Masui, 1982).
Lohka also found that sperm nuclei were immediately
transformed into metaphase chromosomes, instead of
pronuclei, when they were incubated in extracts of CSFarrested unfertilized eggs prepared in a way similar to
that described above but to which the Ca-chelating
agent, EGTA, had been added to prevent egg activation.
However, when Ca ions were added to these extracts to
inactivate MPF and CSF, chromosomes were transformed into interphase nuclei. These results of in vitro experiments demonstrated that MPF and CSF are essential
for the formation and maintenance of metaphase
chromosomes (Lohka and Masui, 1984a). He also confirmed the conclusion from in vivo experiments that activation and inactivation of MPF cause the transition
from interphase to metaphase, and metaphase to interphase, respectively.
However, when sperm nuclei were incubated in the
activated egg cytosols prepared by high speed centrifugation, therefore having cytoplasmic membrane vesicles removed, they failed to form a pronucleus and
never underwent cell cycle changes (Lohka and Masui,
1983; 1984b). Similarly, nuclei incubated in CSF-arrested egg extracts lacking membrane vesicles prepared
by high speed centrifugation also failed to condense
chromosomes to a metaphase state even though high
MPF and CSF activities were detected in the extracts as
mentioned before. Thus, sperm nuclear transformations,
both chromosome condensation and pronuclear formation, were found to be regulated not only by changing
MPF activities in the cytosol, but also by the activities
of cytoplasmic membranes.
In 1985, the cell-free system was developed by Lohka
using Xenopus eggs, after he left Toronto in 1984 to join
Maller’s lab at the University of Colorado as a postdoc.
With this system, they showed that sperm pronuclei
formed in activated egg extracts could be immediately
transformed into metaphase chromosomes when par-
14
tially purified MPF was added (Lohka and Maller,
1985). This procedure was later used for rapid MPF assay for its purification (Lohka et al., 1988). This cell-free
system was further refined by Murray in Kirschner’s lab
at the University of California at San Francisco, and in
1989, they reported that sperm pronuclei formed in this
new cell-free system, called ‘‘cycling extract’’, could perform up to a few more complete cell cycles (Murray and
Kirschner, 1989).
The molecular nature of MPF and CSF
In 1980, Rosenthal et al. found novel proteins that appear during meiotic and mitotic cycles in eggs of the
clam, Spisula, and in 1983, Evans et al., observing that
some of them were synthesized during interphase and
were rapidly degraded at the onset of cell division,
named these proteins ‘‘cyclins’’. The observation that the
cyclin cycle is closely correlated with the chromosome
cycle (Cornall et al., 1983) suggested a close correlation
between MPF activity and the level of cyclin. In 1986,
cyclin genes were cloned from Spisula and sea urchin
eggs, and cyclin A mRNA was injected into Xenopus
oocytes by Swenson et al. (1986). They showed that cyclin mRNA did induce GVBD and chromosome condensation to a metaphase state in the Xenopus oocytes.
By 1986, the gene required for completion of mitosis
in the yeast cell, cdc2, was cloned and sequenced, and
its product was identified as a 34 kD protein kinase
(Hindley and Phear, 1984; Simanis and Nurse, 1986). In
the area of oocyte maturation studies, efforts had been
made to purify MPF. Finally, in 1988, Manfred Lohka
and his associates succeeded in purifying MPF 3000-fold
using in vitro sperm chromosome condensation as the
assay for MPF in Xenopus egg cytosol fractions. They
found that MPF was a phosphoprotein molecule consisting of 32 and 45 kD peptides and capable of phosphorylating its own 45 kD peptide as well as histone H1
in the presence of ATP. In the following few years, it was
found that the 32 kD protein and 45 kD peptide of the
purified MPF cross-reacted with anti-cdc 2 antibody
and the anti-cyclin B antibody, respectively. Thus, MPF
was identified as a protein kinase consisting of two subunits, cdc2 and cyclin B (Nurse, 1990, for a review).
Unfortunately, purification of 1 æCSF has not been
successful to date because of the extreme instability of
its activity in frog-egg extracts. However, its identity was
revealed from a different angle. In 1989, Sagata and his
associates found striking similarities in behaviour between 1 æCSF and Mos, the protein kinase coded by the
proto-oncogene c-mos of 39 kD designated pp39mos.Xe.
In Xenopus oocytes, Mos protein rapidly increases during maturation, reaching a maximum level at M II, but
it rapidly disappears following fertilisation. Mos
mRNA, like 1 æCSF, when injected into cleaving blastomeres of 2-cell embryos arrests the recipient blastomeres
at metaphase. It was also shown that immuno-precipitation of proteins in unfertilised egg cytosols with Mosspecific antibodies deprived the cytosols of CSF activity,
and that centrifugation of the cytosols at 150,000 ¿ g
for 6 h precipitated both Mos protein and CSF activity.
Further, both CSF and Mos were destroyed when Ca
ions were added to the cytosols as well as when Xenopus
eggs were activated by Ca ionophore A23187, indicating
that Mos is as sensitive as 1 æCSF to Ca ions and is destroyed under the same cytoplasmic conditions as those
brought about by egg activation (Watanabe et al., 1991).
Thus, they concluded that ‘‘the c-mos proto-oncogene
product, pp39mos (Mos), is either the long-known endogenous meiotic inhibitor of amphibian eggs, the cytostatic
factor, or at least a catalytic component of it’’ (Sagata
et al., 1989; p. 516).
In 1989, Murray and Kirschner demonstrated that cyclin B and MPF levels both cycled in Xenopus cycling
extracts and that the appearance of MPF and cyclin B
were suppressed when protein synthesis in the extracts
was inhibited or mRNAs were depleted. However, MPF
could appear and cell cycle events could continue if cyclin mRNA was translated even in extracts from which
all endogenous mRNAs had been depleted. If cyclin B
mRNA lacking protease-reactive sequence was translated in extracts, MPF and cyclin B levels stayed high
and failed to cycle. These results clearly proved that progression of cell cycle events in frog egg extracts requires
only synthesis and proteolytic degradation of cyclin B
to cause a rise and fall of MPF activity.
These experiments opened the gate for the molecular
biological approach to the cell cycle control, which has
grown at an amazing speed in the past decade, increasing our knowledge of the regulatory mechanisms of the
cell cycle at the molecular level tremendously. However,
we must note that the old questions about oocyte maturation and egg activation raised in the early years of our
study are still waiting for answers. We have not fully
understood the mechanisms of how maturation-inducing hormones, such as progesterone and 1-MA, act
on the oocyte to give rise to MPF. We still do not know
about the receptor of these hormones in the oocyte or
protein phosphorylation cascades that lead to the appearance of MPF and roles of ions and cyclic nucleotides in the regulation of these processes. In addition,
we have little knowledge about the mechanisms of how
MPF could lead condensation of chromosomes to a
metaphase state and how CSF maintains M phase (Masui and Clarke, 1979; Masui, 1992; 2000; Maller, 1998
for reviews). History tells us that learning from oocyte
maturation and egg activation would help us again to
gain new insight into the regulation of the cell cycle.
Acknowledgements The author thanks Professor Michael Filosa,
Department of Zoology, University of Toronto, for his careful reading of the manuscript and for his kind comments, Mrs. Stacey
Hayden for her reading of the manuscript and editorial assistance,
and Ms. Tammy Hong for preparing diagrams.
15
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