REVIEW ARTICLE

Journal of
592
Cellular
Physiology
Meiotic Cell Cycle Arrest in
Mammalian Oocytes
ANIMA TRIPATHI, K.V. PREM KUMAR, AND SHAIL K. CHAUBE*
Cell Physiology Laboratory, Biochemistry Unit, Department of Zoology, Banaras Hindu University, Varanasi, India

Meiotic cell cycle in mammalian oocytes is a dynamic process that involves several stop/go channels. The cell cycle arrest in oocyte occurs
at various stages such as diplotene, metaphase-I (M-I), metaphase-II (M-II), and so called metaphase-like arrest (M-III). Leutinizing hormone
surge induces meiotic resumption from diplotene arrest in follicular microenvironment by overriding several factors responsible for the
maintenance of meiotic arrest. The inhibitory factors are synthesized in oocyte or in the associated follicular somatic cells and transferred
to the oocyte. The major factors include hypoxanthine, cyclic adenosine 30 , 50 -monophosphate, cyclic guanosine 30 , 50 -monophosphate,
reactive oxygen species, protein kinase A, and protein kinase C. In the presence of active protein kinases, epidermal-like growth factors
are produced that activate mitogen-activated protein kinase in cumulus granulosa cells. The maturation promoting factor, cytostatic
factors, and spindle assembly checkpoint proteins are also involved in the maintenance of arrest at various stages of meiotic cell cycle in
mammalian oocytes. In this review, we briefly summarize the role of these factors in the maintenance of meiotic cell cycle arrest in
mammalian oocytes.
J. Cell. Physiol. 223: 592–600, 2010. ß 2010 Wiley-Liss, Inc.

Meiotic cell cycle in mammalian oocytes starts during fetal life
and gets arrested at diplotene stage of first meiotic prophase at
the time of birth. The diplotene arrest in oocyte may last for
several months or years in follicular microenvironment
depending on the mammalian species (Wassarman and
Albertini, 1994; Sirard, 2001; Trounson et al., 2001; Mehlmann,
2005). Meiotic resumption from diplotene arrest is
morphologically characterized by germinal vesicle breakdown
(GVBD). After GVBD, the proper organization of metaphase
spindle by activated mitogen-activated protein kinase (MAPK)
makes the first meiotic division to progress through
metaphase-I (M-I) manifested by extrusion of first polar body,
and then by cytostatic factor (CSF)-mediated maturation
promoting factor (MPF) stabilization, meiotic cell cycle gets
further arrested at metaphase-II (M-II) until fertilization.
However, in the absence of fertilization in some mammalian
species, postovulatory aging induces exit from M-II arrest, and
eggs are further arrested at metaphase-III-like stage so called
M-III arrest (Eppig et al., 2004; Mehlmann, 2005). The purpose
of this review is to summarize data from several laboratories,
including ours, on the factors responsible for the maintenance
of meiotic arrest. We hope that this review may provide
updated molecular mechanisms of meiotic cell cycle arrest in
mammalian oocytes.
Meiotic Cell Cycle Arrest at Diplotene Stage
It is well established that intraoocyte cyclic adenosine
30 ,50 -monophosphate (cAMP) plays an important role in the
maintenance of meiotic arrest at diplotene stage (Mehlmann,
2005). One long-standing hypothesis is that the continuous
transfer of cAMP from cumulus granulosa cells to the oocyte
through gap junctions maintains meiotic arrest (Fig. 1a) (Dekel
et al., 1981; Wassarman and Albertini, 1994; Chaube, 2002;
Webb et al., 2002). However, it has been recently reported that
intraoocyte cAMP level is essential for the maintenance of
meiotic arrest at diplotene stage in mouse oocyte (Fig. 1a)
(Vaccari et al., 2008). Furthermore, cyclic guanosine 30 ,50 -
monophosphate (cGMP) also acts as an important inhibitory
signal and maintains meiotic arrest in mammalian oocyte. The
cGMP passes from associated cumulus granulosa cells through
gap junctions to the oocyte where it inhibits hydrolysis of cAMP
by phosphodiesterase 3A (PDE3A) (Fig. 1b). This inhibition
maintains a high concentration of intraoocyte cAMP and this
blocks meiotic progression (Norris et al., 2009b; Sun et al.,
2009). However, lack of specific inhibitors for the gap junction
in the oocyte has complicated the efforts to clarify their possible
role in the maintenance of meiotic arrest (Mehlmann, 2005).
Growing body of evidences suggest that the oocyte is capable of
generating sufficient amount of cAMP required for the
maintenance of meiotic arrest (Masciarelli et al., 2004; Vaccari
et al., 2008). The resumption of meiosis after the injection of
G-stimulatory (Gs) inhibitory antibody into oocyte suggests the
involvement of Gs-adenylate cyclase mediated production of
cAMP within the oocyte (Mehlmann et al., 2002). The receptors
coupled to Gs and adenylate cyclases have been reported in
mammalian oocytes (Mehlmann et al., 2004; Hinckley et al.,
2005; Ledent et al., 2005). The activation of G-protein coupled
receptors (GPCRs) in the oocyte plasma membrane that
stimulates Gs protein and subsequently adenylate cyclase is
responsible for the generation of cAMP within the oocyte
(Masciarelli et al., 2004; DiLuigi et al., 2008). The expression of
active adenylate cyclases such as adenylate cyclase 3 (ADCY3)
and adenylate cyclase 9 (ADCY9) have been reported in mouse
and rat oocytes. Furthermore, ADCY3-deficient oocytes show
precautious resumption of meiosis, suggesting that cAMP
accumulation within the oocyte is required for the maintenance
of meiotic arrest (Hornera et al., 2003).
Another primary determinant of intraoocyte cAMP level
and thereby of meiotic arrest is the inactivation of major
cAMP–PDEs responsible for degradation of cAMP in oocyte by
hydrolysis. This concept is strengthened by in vitro
observations that the nonselective PDE inhibitors such as
3-isobutyl-1-methyl xanthine, papaverine, theophylline, and
pentoxyfilline inhibit PDE activity and thereby meiotic
resumption (Downs et al., 1989; Chaube, 2001, 2002; Chaube
et al., 2001). The selective PDE3A inhibitors such as cilostamide
and milrinone maintain oocyte meiotic arrest in several
mammalian species (Tsafrifri et al., 1996; Wiersma et al., 1998;
*Correspondence to: Shail K. Chaube, Cell Physiology Laboratory,
Department of Zoology, Banaras Hindu University, Varanasi
221005, India. E-mail: shailchaubey@gmail.com
Received 14 January 2010; Accepted 15 January 2010
Published online in Wiley InterScience
(www.interscience.wiley.com.), 15 March 2010.
DOI: 10.1002/jcp.22108

ß 2 0 1 0 W I L E Y - L I S S , I N C .
 MEIOTIC CELL CYCLE ARREST IN OOCYTE 593

Fig. 1. a: A diplotene-arrested rat oocyte showing the presence ofgerminal vesicle and nucleolus("). Original magnification, 400T. b: Aproposed
model for the maintenance of meiotic arrest at diplotene stage in preovulatory follicle. The cAMP and cGMP produced by cumulus granulosa cells
are transferred to the oocyte via gap junctions. The increased level of cGMP inactivates PDE3A and prevents hydrolysis of oocyte cAMP. The
increased level of oocyte cAMP increases PKA activity. The increased PKA activity inactivates Cdc25B phosphatase and activates Wee1/Myt1
kinase that results in posphorylation of CDK1 and inactivation of MPF. The increased NO level through iNOS-mediated pathway and inactivation of
MPF finally induces meiotic arrest at diplotene stage. [Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]

Mayes and Sirard, 2002; Lindbloom et al., 2008). More recently,
Vaccari et al. (2008) reported that oocytes deficient in PDE3A
do not hydrolyze cAMP, and thereby cAMP accumulates above
the threshold level resulting in maintenance of meiotic arrest.
Taken together, these studies suggest that GPCR3 and PDE3A
are primary determinants of cAMP levels required for the
maintenance of meiotic arrest (Fig. 1b).
Follicular cumulus granulosa cells encircling oocyte are also
responsible for the maintenance of meiotic arrest at diplotene
stage. The inhibitory factor(s) released from theca and
granulosa cells are passed to the oocyte through gap junctions,
which results in the maintenance of meiotic arrest (Sirard and
Bilodeau, 1990; Sirard et al., 1992; De Loos et al., 1994; Richard
and Sirard, 1996a,b). Furthermore, inhibitory factor produced
by the theca cells also has a short-term effect, but its continued
ability to maintain the oocytes in arrest of meiosis requires
either replenishing every 4 h or continuous presence of theca
cells (Richard and Sirard, 1996b). One of the active inhibitory
factors that have been identified is hypoxanthine (Sirard and
First, 1988; Wassarman and Albertini, 1994). The inhibitory
factor produced by the theca cells requires the cumulus cells to
be in direct contact with the oocyte as the cumulus cells play a
key role in either transporting or processing the inhibitory
factor (Richard and Sirard, 1996a). Incubation of cumulus
oocyte complexes in the presence of theca cell monolayers and
PDE3A inhibitors produces higher rate of meiotic arrest than
those obtained by incubating the cumulus oocyte complexes in
the presence of untreated theca cell monolayers. These findings
suggest that the purines produced by theca cell monolayers act
upstream from the site of action of PDE3A inhibition (Mayes
and Sirard, 2002). Thus, purines released from theca cells
maintain meiotic arrest by inhibiting PDE3A activity and by
increasing intracellular cAMP level (Eppig and Downs, 1984;
Mehlmann, 2005). The increased intraoocyte cAMP level
through discrete set of steps inactivates MPF resulting in the
maintenance of meiotic arrest at diplotene stage of mammalian
oocytes (Wassarman and Albertini, 1994).
MPF plays a key role in maintenance of diplotene arrest. MPF
is a serine–threonine kinase protein heterodimer composed of
a catalytic subunit, cyclin-dependent kinase 1 (CDK1), and a
regulatory subunit, cyclin B (Fig. 1b). The activated form of MPF
involves dephosphorylation at Thr14 and Tyr15 of CDK1 and
its association with cyclin B (Clarke and Karsenti, 1991). In
addition to this, a change in the level of cyclin B by synthesis and
degradation is of central importance for MPF activity (Ledan
et al., 2001). Cyclin B is accumulated in diplotene-arrested
oocytes due to the presence of early mitotic inhibitor1 (Emi1),
an inhibitor of anaphase promoting complex/cyclosome (APC/
C), which is responsible for the destruction of cyclin B
(Marangos et al., 2007). The APC/C is a large assembly of
proteins that associate with one of at least two adopters, Cdc20
and Cdh1, to direct polyubiquitylation of securine, cyclin B, and
other cell cycle regulators for subsequent degradation by the
proteosome. In diplotene-arrested oocytes, high
concentrations of cAMP activate protein kinase A (PKA), and
activated PKA phosphorylates two CDK1 regulators such as
Cdc25B phosphatase (Pirino et al., 2009) and Wee1/Myt1
kinase (Standford and Ruderman, 2005; Han and Conti, 2006).
The inactivation of Cdc25B and activation of Wee1/Myt1 kinase
ultimately inactivate MPF leading to the maintenance of meiotic
arrest in several mammalian species (Han and Conti, 2006;
Potapova et al., 2009).
Recent studies indicate the involvement of reactive nitrogen
species (RNS), such as nitric oxide (NO), in the maintenance of
meiotic arrest at diplotene stage (Nakamura et al., 2002; Sela-
Abramovich et al., 2008). This hypothesis has been supported
by our recent findings that diplotene-arrested oocyte showed
highest inducible nitric oxide synthase (iNOS) expression as
compared to the oocytes arrested at M-I and M-II stages of
meiotic cell cycle (Chaube et al., 2009). Similarly, the highest

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594 TRIPATHI ET AL.

intracellular NO level is associated with diplotene-arrested
oocyte as compared to M-I and M-II stages (Tripathi et al.,
2009). These findings indicate that oocyte is capable of
generating sufficient amount of NO through iNOS-mediated
pathway required for maintenance of meiotic arrested at
diplotene stage in mammalian oocyte (Fig. 1b).
Exit from diplotene-arrest (Fig. 2) is induced in response to
pituitary gonadotropins particularly luteinizing hormone (LH)
(Eppig et al., 2004; Mehlmann, 2005). Stimulation of meiotic
resumption by LH occurs indirectly via its action on the
surrounding granulosa cells rather than on the oocyte itself
since oocyte lacks gonadotropin receptor (Amsterdam et al.,
1975; Dekel, 1988; Zhang et al., 2009). LH binds to its receptor
on mural granulosa cells and initiates signaling pathways
involving cAMP and cGMP as second messengers (Dekel et al.,
1984; Dekel, 1988; Eppig, 1989; Byskov et al., 1997; Liang et al.,
2005; Zhang et al., 2009). In response to LH surge, an increased
cAMP level induces PKA and PKC pathways to produce
epidermal-like growth factors (EGF-like factors) such as
amphiregulin and epiregulin in mural granulosa cells (Park et al.,
2004; Mehlmann, 2005; Chen et al., 2008). These EGF-like
factors, via their receptors on cumulus granulosa cells, induce
MAPK activity (Gall et al., 2005; Mehlmann, 2005; Li et al., 2008).
Studies suggest that MAPK activation in cumulus granulosa cells,
but not in the oocyte, is indispensible for meiotic resumption,
while intraoocyte MAPK activation cascade is required for
postdiplotene event such as meiotic spindle organization (Fan
and Sun, 2004; Liang et al., 2007; Yu et al., 2007; Xiong et al.,
2008; Fan et al., 2009). The activated MAPK interrupts cell-to-
cell communication among cumulus granulosa cells and
between cumulus granulosa cells and oocyte through
phosphorylation of connexion 43 at serine and threonine
residues resulting in the closure of gap junction (Sela-
Abramovich et al., 2005; Norris et al., 2009a; Sun et al., 2009).
The disruption of gap junctions arrests the supply of cAMP from
somatic cells to the oocyte leading to decrease in intraoocyte
cAMP level (Dekel, 1988; Zhang et al., 2009). The disruption of
gap junctions also reduces the transfer of cGMP from cumulus
granulosa cells to the oocyte (Fig. 2). Reduction in the level of
intraoocyte cGMP level results in the activation of PDE3A
activity that further reduces intraoocyte cAMP level. This
possibility is supported by the observation that cGMP maintains
meiotic arrest by inhibiting oocyte PDE3A activity to maintain
cAMP level in the oocyte (Tornell et al., 1991; Wang et al.,
2008).
The net reduction of cAMP within the oocyte inactivates
PKA since high cAMP level and PKA activation play a critical role
in the maintenance of meiotic arrest (Han and Conti, 2006).
In the absence of active PKA, phosphatase such as Cdc25B is not
phosphorylated and remains in its active form. Similarly, Wee1/
Myt1 kinases remain in dephosphorylated and inactive form
(Eppig et al., 2004; Wang et al., 2005; Han and Conti, 2006;
Liang et al., 2007; Solc et al., 2008). Active Cdc25B
dephosphorylates CDK1 (a catalytic subunit of MPF) at Thr14
and Tyr15 on the MPF complex that is maintained by Wee1B
phosphorylation. As a result, CDK1 of MPF complex gets
activated, and meiosis is resumed from diplotene arrest
(Duckworth et al., 2002; Han and Conti, 2006). This possibility
is further supported by the observations that oocytes from
Cdc25B
/
mice remain at GV stage if cultured in vitro and
microinjection of wild-type Cdc25B mRNA triggers the
resumption of meiosis (Lincoln et al., 2002). The cyclin B (a
regulatory subunit of MPF) is accumulated due to Emi1-
dependent inhibition of APCcdh1 in diplotene-arrested oocytes
(Fig. 2). During LH-induced meiotic resumption, Emi1
destruction is stimulated that results in resumption from
diplotene-arrest (Marangos et al., 2007). An exit from
diplotene-arrest can also be achieved if preovulatory oocytes
are removed from its follicular microenvironment that
maintains meiotic arrest by synthesis and secretions of several
inhibitory factors and further cultured under in vitro conditions
(Wassarman and Albertini, 1994; Mehlmann, 2005).

Fig. 2. A proposed model for LH-induced meiotic resumption in follicular oocyte. LH binds to its receptor in mural granulosa cells and induces
production of cAMP through adenylate cyclse (AC) pathway. The increased level of cAMP then stimulates PKA/PKC activity and thereby the
production of EGF-like factors in cumulus granulosa cells. The EGF-like factors induce MAPK activity that disrupts gap junctions among cumulus
granulosa cells and between cumulus granulosa cells and oocyte. The interruption of communication between cumulus granulosa cells and oocyte
blocks the transfer of cAMP and cGMP. The reduced level of cGMP activates PDE3A that further reduces cAMP level by hydrolysis, which is
generated by oocyte itself through GPR3/AC pathway. The net reduction of oocyte cAMP reduces oocyte PKA activity. The reduced PKA activity
activates Cdc25B phosphatase and inactivates Wee1/Myt1 kinase that results in deposphorylation of CDK1 and activation of MPF. The active MPF
finally induces resumption of meiosis from diplotene arrest. [Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]

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 MEIOTIC CELL CYCLE ARREST IN OOCYTE
Meiotic Cell Cycle Arrest at M-I Stage
Meiotic cell cycle in mammalian oocytes remains arrested at M-I
stage (Fig. 3a) until all sister chromatids have been properly
attached to the bipolar spindle and are aligned at the metaphase
plate (Wassmann et al., 2003). The spindle assembly checkpoint
(SAC) proteins ensure the correct segregation of homologous
chromosomes and provoke a cell cycle arrest in metaphase if any
chromosome is not correctly attached to the bipolar spindle
(Niault et al., 2007). Functional SAC exists during M-I stage in
mouse oocytes and acts through their checkpoint proteins such
as Mad2, Bub1, and Bub3 (Fig. 3b) (Wassmann et al., 2003; Li et al.,
2009). The Mad2 is present at kinetochore during early M-I
(Hupalowska et al., 2008). Over-expression of Mad2 leads to cell
cycle arrest at M-I stage, but the expression of a dominant
negative Mad2 protein interferes with the proper spindle
checkpoint arrest (Wassmann et al., 2003). The SAC proteins do
not control the timing of M-I but rather delay metaphase–
anaphase transition until the spindle microtubules are attached
to kinetochore and chromosomes are properly aligned on the
metaphase plate (Brunet and Maro, 2005). The APC/C is the only
known target of the SAC proteins (Fig. 3b).
The SAC proteins ensure accurate chromosome segregation
by inhibiting APC/C and delaying anaphase onset (Brunet and
Maro, 2005). The Mad2 directly interacts with APC/C adopter
Cdc20 and inhibits APC/C activation for correct separation of
homologous chromosome during first meiotic division (Fig. 3b).
(Wassmann et al., 2003). Similarly, Bub1 and Bub3 are also
found to interact with Cdc20 of APC/C, but whether they
interact in the same complex as Mad2 or in a complex apart
need to be clarified (Wassmann et al., 2003). Formation of
functional spindle correlates with the progressive increase in
MPF activity (Madgwick et al., 2004). The correct positioning of
spindle requires MPF activity, while spindle migration and APC/
C activation are initiated once MPF activity reaches higher level
(Brunet and Maro, 2005). The cyclin B levels through the
regulation of MPF activity synchronize the different events of
spindle formation, spindle migration, and APC/C activation
(Brunet and Maro, 2005). This possibility is further supported
Fig. 3. a: A M-I-arrested rat oocyte showing germinal vesicle breakdown. b: A proposed model for meiotic arrest at M-I stage in preovulatory
follicle. The spindle assembly checkpoint (SAC) proteins such as Mad2, Bub1, and Bub3 directly interact with APC/C adopter Cdc20 and inhibit
APC/C activity which then stabilizes MPF. Activation of Mos/MEK/MAPK/Rsk90 pathway also stabilizes MPF. The stabilization of MPF and
generation of ROS maintain meiotic arrest at M-I stage. [Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]
JOURNAL OF CELLULAR PHYSIOLOGY
595
by the observation that the depletion of Emi1 in GV-arrested
oocytes increases the cyclin B1 destruction, resulting in an
attenuation of MPF activation and a delay of entry into M-I stage
(Marangos and Carroll, 2004; Marangos et al., 2007). Although
reduction in the level of Emi1 is required for the resumption of
meiosis from diplotene-arrest, its low level is sufficient to
accelerate entry into M-I stage, spindle formation, and
progression through M-I (Marangos et al., 2007).
Activation of Mos/MAPK also plays a significant role in
microtubule reorganization and in positioning of metaphase
spindle to the oocyte cortex, an essential event for metaphase
to anaphase transition (Zhou et al., 1991; Verlhac et al., 1994,
1996; Choi et al., 1996a). In the absence of Mos/MAPK
activation, the extensive astral-like prophase microtubule
arrays appear to persist that may prevent proper association of
the spindle with the inner surface of the oocyte cortex (Choi
et al., 1996b). Therefore, metaphase spindle fails to properly
translocate to the cell surface causing abnormal formation of
the first polar body, and oocyte remain arrested at M-I stage
(Choi et al., 1996a,b; Araki et al., 1996; Liang et al., 2007).
Growing body of evidences suggest that reactive oxygen
species (ROS) may be involved in the maintenance of M-I arrest
in mammalian oocyte. This possibility is strengthened by the
observations that supplementation of exogenous H2O2 inhibits
first polar body emission in rat and mice oocytes cultured in
vitro (Chaube et al., 2005; Tamura et al., 2008). Recently,
we have reported that the intracellular level of H2O2 in
M-I-arrested oocyte is higher as compared to diplotene-
arrested oocyte (Tripathi et al., 2009). The possibility exists
that the M-I-arrested oocytes generate H2O2 through
mitochondrial pathway leading to increased intracellular H2O2
level and meiotic cell cycle arrest in oocytes (Goud et al., 2008).
The possible mechanism by which ROS maintains meiotic arrest
is yet to be clarified.
Meiotic Cell Cycle Arrest at M-II Stage
The stabilization of MPF activity and proper organization of
the spindle by specific mechanisms are required for the
596 TRIPATHI ET AL.

Fig. 4. a: M-II-arrested oocyte showing presence of first polar body (").Original magnification, 400T. b: A proposed model for maintenance of
meiotic arrest at M-II stage. The c-mos/MEK/MAPK pathway directly induces MPF stabilization, and indirectly this pathway recruits PP2A to
activate Emi2. The activated Emi2 then forms a complex with APC/C through its adopter Cdc20 leading to the stabilization of MPF. c: Mos/MEK/
MAPK pathway using two proteins such as MISS and DOC1R induces spindle stability, and cyclin B synthesis induces MPF stabilization. The spindle
stability and MPF stabilization induce maintenance of meiotic arrest at M-II stage. [Color figure can be viewed in the online issue, which is available
at www.interscience.wiley.com.]

maintenance of M-II arrest in the mammalian eggs (Fig. 4a). The
stabilization of MPF is maintained by CSF and by Mos-mediated
MAPK pathway (Shoji et al., 2006; Madgwick and Jones, 2007;
Perry and Verlhac, 2008). The CSF by definition does not
describe a single molecule or protein, but rather an activity
found in the egg (Wu and Kornbluth, 2008). Members of the
novel Emi/Erp family of protein have been reported as
important components of CSF (Schmidt et al., 2006). The
Emi/Erp family comprises only two known members, that is,
Emi1 and Emi2 (Schmidt et al., 2006). Emi1 plays an important
role in timing of the early stages of meiotic maturation before
handing over to SAC proteins in M-I. The arrest at M-II has been
recently attributed to the Emi1 orthologue Emi2 (Hansen et al.,
2006; Shoji et al., 2006; Madgwick and Jones, 2007). The Emi2
CSF pathway ultimately inhibits an ubiquitin ligase called APC/C
(Schmidt et al., 2006; Tang et al., 2008).
The APC adopter such as Cdh1 is abundant in deplotene-
arrested oocytes but declines sharply in M-II-arrested oocytes.
On the other hand, Cdc20 whose protein level is low in
deplotene-arrested oocytes increases in M-II-arrested oocytes
(Shoji et al., 2006). Hence, Cdc20 plays a role as a sole abundant
APC/C adopter in CSF-arrested oocytes (Shoji et al., 2006). For
the maintenance of M-II arrest, complex of Emi2 with APC/C
adopter Cdc20 is essential (Fig. 4b) (Liu et al., 2006). On the
other hand, release of Cdc20 from Emi2 and its subsequent
binding with APC/C results in its activation. This activation is
achieved either in the presence of fertilizing spermatozoa or by
parthenogenetic agents that cause calcium release from internal
stores. An increase in intracellular calcium level activates
calcium/calmodulin-dependent PK-II (CamKII) in the egg. The
activated CamKII causes phosphorylation of Emi2. The
phosphorylation of Emi2 is further potentiated by another

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 MEIOTIC CELL CYCLE ARREST IN OOCYTE
polo-like-kinase resulting in complete inactivation and
degradation of Emi2 (Hansen et al., 2006, 2007; Madgwick and
Jones, 2007). The destruction of Emi2 results in the activation of
APC/C that leads to exit from M-II arrest (Masui and Markert
1971; Masui, 2001; Runft et al., 2002; Hansen et al., 2006; Shoji
et al., 2006; Madgwick and Jones, 2007).Another group of
proteins that include Mos (pp39 mos), MAPK kinase kinase
(MEK), and MAPK are involved in the maintenance but not in
the establishment of M-II arrest in mammalian oocytes (Ito et al.,
2007; Madgwick and Jones, 2007). The c-mos, a protooncogene
from a family of kinases that functions as a signal transduction
molecule, has been reported to activate MEK. The MEK serves
as the upstream activator of MAPK. The MAPK is an upstream
activator of ribosomal protein S6 kinase (p90Rsk) that induces
SAC protein activation and thereby inhibition of APC/C (Maller
et al., 2002; Madgwick and Jones, 2007). Although p90Rsk and
SAC proteins play no essential role during CSF arrest in
mammalian eggs, the role of SAC proteins in coordination with
microfilament dynamics and meiotic exit has been reported
(Shoji et al., 2006). In non-mammalian vertebrates, Cdc2/cyclin
B phosphorylates Emi2, weakens Emi2-APC/C interaction,
promoting dissociation of APC/C that leads to exit from M-II
arrest. However, p90Rsk acts downstream to MAPK in Mos
pathway and recruits protein phosphatase 2A (PP2A) to Emi2
causing dephosphorylation of Emi2. As a result,
dephosphorylated Emi2 does not dissociate from APC/C
complex and the CSF arrest is maintained (Wu and Kornbluth,
2008). However, eggs collected from Rsk-knockout mouse still
maintain a CSF arrest suggesting the involvement of a redundant
pathway that is responsible for Emi2 phosphorylation and PP2A
binding (Dumont et al., 2005; Tang et al., 2008; Wu and
Kornbluth, 2008).
For the maintenance of M-II arrest in the mammalian eggs,
proper organization of the spindle by specific mechanisms is
required. The MAPK interacting and spindle stabilizing (MISS)
and DOC1R (deleted in oral cancer one related, a murine
homologue of a potential human tumor suppressor gene) are
two MAP kinase substrates associated with the spindle in
M-II-arrested oocytes (Fig. 4c) (Lefebvre et al., 2002; Terret
et al., 2003). The DOC1R accumulates during meiotic
maturation, while MISS is present only during M-II. In the
absence of these two proteins, the M-II spindle forms normally,
but later become disorganized indicating a role for both
proteins in the maintenance of the spindle structure during the
arrest (Brunet and Maro, 2005). MISS and DOC1R are
regulated by multiple phosphorylations through the activity of
MAP kinase and MPF. Thus, cooperation between MPF and
MAP kinase pathway (leading to CSF activity) is at play to
maintain the spindle structure when the metaphase stage is
highly prolonged (Brunet and Maro, 2005).
The balanced synthesis and degradation of cyclin B also plays
a key role during M-II arrest (Kubiak et al., 1993). The
equilibrium between these two processes is dependent upon
CSF that slows down degradation (Kubiak et al., 1993) and
continuous synthesis of cyclin B that is maintained at the highest
level (Winston, 1997). Cyclin B degradation occurs once
spindle formation has been completed and the chromosome
aligned on the metaphase plate in M-II-arrested oocyte (Brunet
and Maro, 2005). During CSF arrest, the SAC is inactive and
neither Bub1 nor Mad2 is required for M-II arrest (Tsurumi
et al., 2004; Dumont et al., 2005; Perry and Verlhac, 2008).
Thus, APC/C inhibitor responsible for the maintenance of
higher level of MPF activity during the M-II arrest remains
unidentified.
The role of ROS in the maintenance of M-II arrest in
mammalian oocyte is ill understood. However, recent findings
from our laboratory indicate that the intracellular level of H2O2
in M-II-arrested oocyte is higher as compared to diplotene-
arrested oocyte (Tripathi et al., 2009). The possibility exists that
JOURNAL OF CELLULAR PHYSIOLOGY
597
the generation of H2O2 through mitochondrial pathway (Goud
et al., 2008a) might be associated with the maintenance of M-II
arrest. Further studies are required to explore the possible
mechanism underlying H2O2 generation and its involvement
during M-II arrest in mammalian oocyte.
Meiotic Cell Cycle Arrest at M-III-Like Stage
In most of the mammalian species, ovulated eggs are arrested at
M-II stage of meiotic cell cycle with first polar body. Egg
activation that includes exit from M-II arrest and extrusion of
second polar body is generally associated with the transient
increase of intracellular calcium ion [Ca2þ]i at the time of
fertilization (Kline and Kline, 1992; Xu et al., 1997; Raz et al.,
1998; Whitaker, 2006). In the absence of fertilization,
postovulatory aging mimics the action of fertilizing
spermatozoa, increases [Ca2þ]i, and induces exit from M-II
arrest (Vincent et al., 1992; Xu et al., 1997; Raz et al., 1998).
These partially activated eggs do not progress further and get
arrested again in a new metaphase-like stage called metaphase-
III (M-III) in few mammalian species (Fig. 5a) such as mouse
(Vincent et al., 1992; Xu et al., 1997), hamster (Austin, 1956),
and rat (Zernika-Goetz, 1991; Raz et al., 1998; Ross et al., 2006;
Chaube et al., 2007; Galat et al., 2007; Tamura et al., 2008). The
detail molecular mechanism(s) underlying M-III arrest is still ill
understood. However, one of the important factors
responsible for M-III arrest in aged eggs might be the insufficient
release of cytosolic calcium from internal stores like
mitochondria and endoplasmic reticulum. Due to insufficient
calcium release, Cdc2 kinase activity is not fully re-established in
M-III-arrested eggs as compared to M-II-arrested eggs (Kubiak
et al., 1992). In M-III-arrested eggs, sufficient CSF activity is still
present to stabilize residual or newly formed MPF activity
resulting in M-III arrest in few mammalian species (Kubiak et al.,
1992; Vincent et al., 1992). Due to the presence of CSF activity,
chromatids remain condensed and microtubular metaphase
spindle reforms in M-III-arrested eggs (Kubiak et al., 1992;
Vincent et al., 1992). Two unknown proteins of 133- and 138-
kDa are present in M-III-arrested eggs and their reduction may
induce parthenogenetic activation indicating the importance of
these two proteins in the maintenance of M-III arrest (Fig. 5b)
(Liu et al., 1998).
Recently, a novel and unique role of H2O2 in the
enhancement of postovulatory aging has been reported (Goud
et al., 2008). Similarly, depriving the oocytes of NO results in
the deterioration of oocyte quality postovulation (Goud et al.,
2005). A possibility exists that the intracellular H2O2 which is
generated through mitochondrial pathway (Goud et al., 2008)
might be involved in the maintenance of M-III arrest in
mammalian oocytes. However, the mechanism by which H2O2
generation leads to M-III arrest remains to be elucidated.
In human, an ovarian stimulation protocol generates
predominantly M-II-arrested eggs with first polar body, but a
small proportion (10–15%) of oocytes are also obtained that
are arrested either at diplotene or at M-I stages of meiotic cell
cycle (Bergere et al., 2001; Levran et al., 2002; Mrazek and Fulka,
2003). In some IVF patients, M-II stage oocytes were found to
contain two pronuclei without extrusion of second polar body.
In some other cases, extrusion of second polar body did not
occur, although multiple pronucelei were detected (Levran
et al., 2002; Miyara et al., 2003). In addition, in few mammalian
species including rat and mice, ovulated eggs extrude second
polar body and further arrested at M-III stage of meiotic cell
cycle (Chaube et al., 2008; Tripathi et al., 2009). This is the
typical situation for the oocytes that are unable to complete
meiotic cell cycle and are arrested at various stages of meiotic
cell cycle which may be treated as pathological arrest. The
pathological arrest at various stages of meiotic cell cycle might
be either due to the deficiency of one or other factor(s)
598 TRIPATHI ET AL.
Fig. 5. a: The M-III-arrested oocyte showing degenerating first polar body and extrusion of second polar body ("). Original magnification, 400T.
b: A proposed model for the maintenance of meiotic arrest at M-III stage in egg. Postovulatory aging induces the release of insufficient cytosolic
calcium ion. The presence of insufficient calcium results in the incomplete destruction of CSF leading to MPF stabilization. Two unidentified
proteins of 133 and 138 kDa and ROS throughunknown pathway induce M-III arrest. [Color figure can be viewed in the online issue, which isavailable
at www.interscience.wiley.com.]
responsible for resumption of meiosis or due to increased level
of one or more than one factor(s) required for the maintenance
of meiotic arrest as explained in this review article.
Conclusion
Several factors are involved in the establishment and
maintenance of meiotic arrest in mammalian oocytes including
human. The cAMP and hypoxanthine are released from somatic
cells to maintain diplotene-arrest for a long time in follicular
microenvironment. The cAMP is continuously produced from
cumulus granulosa cells and transferred to the oocyte via gap
junctions. Oocyte is also capable of accumulating cAMP either
by activation of ADCY3 or by inactivation of PDE3A. Increased
intraoocyte cAMP inactivates MPF through PKA/Cdc25B/
CDK1 pathway and thereby maintains meiotic arrest. The CSFs
maintain high level of MPF by inhibiting cyclin B degradation
through APC/C–ubiquitin-mediated proteolysis.The
accumulation of cylin B and its association with Cdk1 results in
MPF stabilization. MPF is also stabilized through Mos/MEK/
MAPK pathway. The MPF stabilization is essential for the
maintenance of meiotic arrest. Abnormal production of SACs
proteins, overproduction of ROS such as H2O2 and RNS such
as NO are also associated with the maintenance of meiotic
arrest. However, the mechanism by which ROS and RNS
maintains meiotic arrest is yet to be investigated. Further
studies in this area may yield important new insights into the
mechanisms regulating meiotic cell cycle arrest in mammalian
oocytes.
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