| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH |
Departments of1 , Embryology2 Cytology, Institute of Zoology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland3 Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK4 Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK
Correspondence should be addressed to A Ajduk; Email: aajduk{at}biol.uw.edu.pl
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
|---|
During the meiotic cycle of mammalian oocytes (commonly known as meiotic maturation) the cyclin B1 levels, and, as a consequence, the MPF activity, fluctuate in a well-defined manner. They are both low in oocytes arrested in the prophase of the first meiotic division (germinal vesicle or GV oocytes) and rise gradually as oocytes resume meiosis, reaching maximum levels in the metaphase of the first meiotic division (metaphase I). At metaphase I/anaphase I transition, an abrupt degradation of cyclin B1 leads to the inactivation of MPF. Then, as the oocytes progress to metaphase of the second meiotic division (metaphase II), cyclin B1 level and MPF activity rise again and remain high during metaphase II arrest (Winston 1997, Ledan et al. 2001, Nixon et al. 2002, Hyslop et al. 2004, Marangos & Carroll 2004). A trigger necessary to terminate metaphase II arrest is provided by a fertilizing spermatozoon, which introduces an oocyte-activating factor, phospholipase C zeta (PLC zeta, Saunders et al. 2002). PLC zeta, via inositol 1,4,5-trisphosphate production, induces Ca2+ oscillations that, in turn, leads to proteosomal degradation of cyclin B1, inactivation of MPF, and completion of meiosis (Nixon et al. 2002, Hyslop et al. 2004).
Under experimental conditions spermatozoa may also penetrate maturing oocytes, i.e., oocytes that have not yet reached metaphase II (Clarke & Masui 1986, 1987, Pyrzynska et al. 1996, Ajduk & Maleszewski 2004). Although it is well known that maturing mouse oocytes react to experimental fertilization or sperm factor injection with attenuated Ca2+ oscillations (lower amplitude and/or number of Ca2+ transients; Carroll et al. 1994, Jones et al. 1995, Cheung et al. 2000, reviewed in Ajduk et al. 2008), the downstream effect of such premature fertilization has not been examined. In the present work, we explore how mono- and polyspermic fertilization of oocytes at different stages of meiotic maturation influences the cyclin B1 level and MPF activity in these cells. Our results confirm that fertilization causes cyclin B1 degradation and long-lasting MPF inactivation only in oocytes that are arrested in metaphase II. We also found that although we did not observe degradation of cyclin B1 in fertilized prometaphase I oocytes, MPF activity transiently decreases in these cells. Therefore, we attempted to elucidate the mechanism that causes the inactivation of MPF in such fertilized prometaphase I oocytes.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
|---|
|
Spermatozoa were added to the maturing oocytes 
2 h after GVBD; however, fertilization occurred at different times, i.e., either in prometaphase I, or metaphase I or metaphase II. Thus, the precise moment of fertilization was estimated by the time of the first Ca2+ transient. Since a membrane block to polyspermy develops in fertilized oocytes in 1 h (Maluchnik & Borsuk 1994), we assumed that in polyspermic oocytes all spermatozoa fused at the similar time. In all examined oocytes penetrated in prometaphase I by a single spermatozoon (7/7) or several spermatozoa (7/7), Ca2+ oscillations caused by sperm penetration did not visibly affect the increase in cyclin B1-luc (Fig. 2A and B). This observation was confirmed by the analysis of the rate of cyclin B1-luc increase in mono- and polyspermic oocytes, as well as in unfertilized ones. To this point, we calculated an inclination angle of the rising slope of cyclin B1-luc curve (Fig. 2A). We found that, according to the tangent values of the inclination angles, the cyclin B1-luc level in fertilized (by one or more sperm) and unfertilized oocytes rose at the same rate (Table 1). Similarly, in all oocytes fertilized in metaphase I, either by a single spermatozoon (17/17) or multiple spermatozoa (6/6), cyclin B1-luc level remained unchanged during Ca2+ oscillations (Fig. 2C and D). In comparison, if fertilization occurred in metaphase II, either in in vitro matured or in freshly ovulated oocytes, in all examined cases (respectively 6/6 and 8/8), a rapid decrease in cyclin B1 level was recorded during Ca2+ oscillations (Fig. 2E and F). Moreover, our recordings clearly indicated that fertilization-induced degradation of cyclin B1 was a continuous, not a stepwise process.
|
|
|
2 h after GVBD. To confirm the validity of our assay conditions, we first measured histone H1 kinase activity (hereinafter called MPF activity) in metaphase II oocytes (16–18 h post-human chorionic gonadotropin (hCG)) in which fertilization triggers the proteolysis of cyclin B1. The mean MPF activity 1, 2, 3, and 4.5 h after fertilization of metaphase II oocytes was, as expected, significantly lower than in control, unfertilized oocytes (P<0.05; Fig. 4A). Intriguingly, a decrease in MPF activity occurred also in maturing oocytes fertilized 2 h after GVBD, even though cyclin B1 was not degraded in such oocytes. However, the observed decrease in MPF activity was transient and less prominent than that occurring in fertilized metaphase II oocytes. Between 1 and 2 h after fertilization of maturing oocytes the MPF activity was significantly decreased compared with control oocytes (respectively 67.8±32.6% and 57.1±33.7% of control activity, P<0.05), whereas 3 h after fertilization the MPF activity in fertilized oocytes had increased to the same level as in unfertilized counterparts (Fig. 4A).
|
To determine whether inactivation of MPF triggered in fertilized maturing oocytes is Ca2+-dependent, we measured the MPF activity in maturing oocytes that had been incubated with a Ca2+ chelator, BAPTA-AM, prior to fertilization. Interestingly, preincubation of maturing oocytes with BAPTA-AM did not prevent the decrease in MPF activity. Two hours after fertilization (three to nine sperms) of BAPTA-AM treated oocytes the MPF activity was 30% lower than in control, unfertilized maturing oocytes (P<0.05; Fig. 4B). In comparison, in fertilized metaphase II oocytes, which were pretreated with BAPTA, the MPF activity remained at the same level as observed in control oocytes (data not shown).
The decrease in MPF activity in polyspermic maturing oocytes was caused by a change in phosphorylation status of CDC2A
Since cyclin B1 was not degraded in maturing oocytes that were fertilized in a polyspermic way, we searched for another reason of MPF inactivation. We investigated whether the observed decrease in MPF activity could be caused by a change in the phosphorylation status of CDC2A. Western blot analysis revealed that the amount of CDC2A phosphorylated on Thr161 (CDC2A-pThr161) was significantly lower in maturing oocytes fertilized by several spermatozoa than in control unfertilized maturing oocytes, whereas the level of total CDC2A was not affected (Fig. 5A). On the other hand, CDC2A phosphorylated on Tyr 15 (CDC2A-pTyr15) was absent in both polyspermic maturing oocytes and their control counterparts. However, the same antibody against CDC2A-pTyr15 detected a clear band corresponding to CDC2A-pTyr15 in a sample prepared from similar number of GV oocytes (Fig. 5C).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
|---|
Fertilization-induced degradation of cyclin B1 requires metaphase arrest of oocytes
We found that Ca2+ oscillations caused by fertilization of maturing oocytes did not lead to a decrease in cyclin B1 level, whereas fertilization of metaphase II oocytes induces rapid degradation of cyclin B1. This result agrees with the data obtained by Marangos & Carroll (2004), who showed that introduction of inositol 1,4,5-trisphosphate triggers proteolysis of cyclin B1 in metaphase II oocytes, but not in maturing ones. Similarly, Hyslop et al. (2004) revealed that ethanol-induced Ca2+ increase was able to affect cyclin B1 levels only in oocytes arrested in metaphase II stage or, in case of abnormal meiosis, in oocytes prematurely arrested in metaphase I. It seems that maturing oocytes lack a mechanism linking fertilization-induced Ca2+ release with degradation of cyclin B1. This mechanism depends on cytostatic factor (CSF) activity, which appears for the first time in oocytes arrested in metaphase II (Kubiak et al. 1993, Nixon et al. 2002, Hyslop et al. 2004, Marangos & Carroll 2004), or, in some rare cases, in oocytes arrested prematurely in metaphase I (Eppig et al. 1994, Ciemerych & Kubiak 1998, Hyslop et al. 2004). CSF stabilizes MPF activity by inhibiting anaphase promoting complex/cyclosome (APC/C)-mediated ubiquitination of cyclin B and in turn, its proteolysis. The level of Emi2, a key element of the CSF activity (Rauh et al. 2005, Tung et al. 2005, Shoji et al. 2006), increases during meiotic maturation of mouse oocytes and reaches its maximum in metaphase II oocytes (Madgwick et al. 2006), i.e., when CSF starts functioning. Emi2 binds to CDC20, the APC/C activator, and prevents activation of APC/C (Shoji et al. 2006). Ca2+ transients caused by fertilization of metaphase II oocytes lead to the degradation of Emi2 (Rauh et al. 2005, Hansen et al. 2006) and in consequence of the activation of APC/C and degradation of cyclin B1 (Madgwick et al. 2006). In maturing oocytes, in which CSF activity is absent, this mechanism does not function.
It also appears that both dissociation of cyclin B1 from CDC2A and its subsequent proteolysis may be prevented in fertilized maturing oocytes by an active spindle assembly checkpoint (SAC). SAC is inactivated when all kinetochores of all chromosomes become stably connected to the spindle microtubules, i.e., just before first meiotic division (Brunet et al. 1999). Until this moment proteins engaged in SAC, such as MAD2L1 (Homer et al. 2005) or BUB1B (Tsurumi et al. 2004), inhibit the APC/C and prevent ubiquitination of cyclin B1 and other crucial M-phase related factors (Nishiyama et al. 2000, Sudakin et al. 2001, Tang et al. 2001, Fang 2002). In the absence of ubiquitination, cyclin B1 can be neither detached from CDC2A nor degraded.
Polyspermic fertilization of maturing oocytes induces dephosphorylation of CDC2A at Thr161 and leads to a transient decrease in MPF activity
Although cyclin B1 was not degraded in fertilized maturing oocytes, we found that the MPF activity in such oocytes was decreased 1 and 2 h after fertilization. A transient decrease in MPF activity is also observed during transition between metaphase I and II, or, in case of suboptimal activation conditions, in transition between metaphase II and III (Kubiak 1989, Kubiak et al. 1992). In both these situations, inactivation of MPF is triggered by degradation of cyclin B1, although separase has been reported recently to act as an additional factor that inhibits CDC2A at the first meiotic division (Gorr et al. 2006). Our results suggest, however, that the decrease in MPF activity in polyspermic maturing oocytes depends on a different mechanism and may be related to the dephosphorylation of CDC2A at Thr161. We did not detect any CDC2A phosphorylated on Tyr15 either in fertilized or control maturing oocytes, but we cannot completely exclude the possibility that the inactivation of MPF was partially caused by an increase in phosphorylation of Tyr15 that was too small to be detected by immunoblotting. Experiments performed on meiotic and mitotic Xenopus extracts confirm that both dephosphorylation of Thr161 and phosphorylation of Tyr15 and Thr14 participate in the process of inactivation of MPF (Lorca et al. 1992, Chesnel et al. 2005, 2007, D'Angiolella et al. 2007).
Our data indicate that the decrease in MPF activity in polyspermic maturing oocytes was an effect of an introduction of additional nuclei that changed the nucleo-cytoplasmic ratio of the oocytes. We showed that, similarly as it happens in polyspermic oocytes, the MPF activity was decreased in prometaphase I oocytes fused with thymocytes. We hypothesize that the additional interphase nuclei provide new substrates for CDC2A–cyclin B1 complex, such as lamins, nucleoporins, etc. (reviewed in Margalit et al. 2005). This in turn may lead to a decrease in the phosphorylation of other CDC2A–cyclin B1 substrates, including cyclin dependent kinase 7 (CDK7), which phosphorylates CDC2A at Thr161 (Solomon et al. 1992, Poon et al. 1993). Decreased phosphorylation compromises the activity of CDK7 (Martinez et al. 1997, Garrett et al. 2001) and, in consequence, may result in lower phosphorylation of CDC2A at Thr161 and inactivation of MPF. CDC25 (a phosphatase responsible for dephosphorylation of CDC2A at Thr14 and Tyr15; Strausfeld et al. 1991) and WEE1 (a kinase that phosphorylates CDC2A on Tyr15; Parker & Piwnica-Worms 1992) are other CDC2A–cyclin B1 substrates. Phosphorylation catalyzed by CDC2A–cyclin B1 activates CDC25 (Hoffmann et al. 1993) and inhibits WEE1 (Mueller et al. 1995). Thus, a decrease in the phosphorylation level of either CDC25 or WEE1 may lead to increased phosphorylation of CDC2A at Tyr15 and the inactivation of MPF. It is probable that the inactivation of MPF can be reversed after some time, when nuclear envelopes of the additional nuclei dissemble and their chromatin condenses.
Oxidative stress leads to dephosphorylation of CDC2A at Thr161 and a decrease in MPF activity
It was tempting to speculate that polyspermic fertilization, which involves introduction of an excessive amount of highly oxidized sperm-specific protamines (reviewed in Balhorn 1982), might have caused a change in the redox state of the maturing oocyte and consequently led to an inhibition of kinases and phosphatases responsible for keeping MPF active (Savitsky & Finkel 2002, Sohn & Rudolph 2003). However, we did not register any decrease in glutathione level in polyspermic maturing oocytes compared with their unfertilized counterparts. Glutathione is the main guardian of a reductive environment in oocytes (reviewed in Luberda 2005) and is responsible for the reduction of protamines introduced by sperm, so its concentration should have been affected if polyspermic fertilization actually led to an oxidative stress.
Even though oxidative stress is not responsible for the inactivation of MPF caused by the polyspermic fertilization, we found that a thiol-oxidizing agent, diamide, inhibited MPF activity in maturing oocytes. Inactivation of MPF caused by an oxidative stress induced by selenium deficiency was reported previously in male germ cells (Kaushal & Bansal 2007). We also determined that diamide treatment led to dephosphorylation of CDC2A at Thr161. Thus, we can speculate that oxidative stress can affect, either in direct or indirect manner, enzymes regulating phosphorylation of CDC2A at Thr161, i.e., CDK7 (Solomon et al. 1992, Poon et al. 1993) and/or protein phosphatase 2C (Cheng et al. 1999, De Smedt et al. 2002). In addition, we cannot completely exclude that diamide affects phosphorylation of CDC2A at Tyr15 in maturing oocytes, but our results do not confirm this possibility. However, it has been shown that an active site of CDC25 contains a cysteine residue, which is highly susceptible to oxidation. Oxidative stress therefore leads to inactivation of CDC25 and consequently may cause an increase in the amount of CDC2A phosphorylated on Thr14 and Tyr 15, and a decrease in MPF activity (Savitsky & Finkel 2002, Sohn & Rudolph 2003).
In summary, our results provided new insights into the mechanism of regulation of MPF activity in mammalian oocytes. We have shown, using cyclin B1 tagged with a firefly luciferase, that degradation of cyclin B1 in fertilized metaphase II oocytes was a continuous, not a stepwise, process. Moreover, we have demonstrated that polyspermic fertilization of prometaphase I oocytes triggers a transient decrease in MPF activity that does not involve proteolysis of cyclin B1. It seems that the observed MPF inactivation was caused, at least in part, by dephosphorylation of CDC2A at Thr161. Our results also indicate that the MPF activity in maturing mouse oocytes can be effectively affected by the nucleo-cytoplasmic ratio.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
|---|
Media and culture conditions
Medium M2 (medium 16 buffered with HEPES; Fulton & Whittingham 1978) containing BSA (4 mg/ml) was used for collection of oocytes and for culture of maturing and ovulated oocytes. Spermatozoa were capacitated in T6 medium (Quinn et al. 1982) or fertilization medium (Fraser 1982) containing BSA (4 mg/ml, Pentex, Bovine Albumin Crystallized; Miles Inc., Kankakee, IL, USA). In vitro fertilization was performed in the fertilization medium or, for the imaging experiment, in HEPES-buffered KSOM (see below). Fertilization medium, as well as T6 medium, was preincubated before use for about 24 h at 37.5 °C in a humidified atmosphere of 5% CO2 in the air. All incubations were carried out in droplets of medium under mineral oil at 37.5 °C in a humidified atmosphere of 5% CO2 in the air.
Oocyte collection and handling
Follicular development was stimulated in 4- to 8-week-old F1 (CBA/HxC57Bl6 or C57Bl6xCBA/H) or MF1 female mice by an i.p. injection of 10 IU of pregnant mares' serum gonadotropin (PMSG, ‘Folligon’; Intervet, Warsaw, Poland). After 46–52 h, females were killed by cervical dislocation. Fully grown oocytes were released from ovarian antral follicles into M2 medium. Oocytes were freed from cumulus cells by pipetting and were cultured in M2 for 1.5 h. After that time, only oocytes that underwent GVBD, i.e., resumed meiosis, were selected and subjected to the experimental procedure.
To induce ovulation, female F1 or MF1 mice were injected with 10 IU of PMSG followed by 10 IU of hCG (‘Chorulon’, Intervet) 46–52 h later. Metaphase II oocytes were collected 16–17 h after hCG injection. Ovulated oocytes surrounded by follicular cells were released from the oviducts into a solution of hyaluronidase (150 IU/ml) in PBS (Biomed, Lublin, Poland). After dispersing of follicular cells, oocytes were washed in M2 and cultured until beginning of the experimental procedure, i.e., for 1.5–2 h.
Depending on the experiment, both maturing and metaphase II oocytes were subjected either to IVF (see below), injection of Ca2+ indicator and cRNA (see below), fusions with thymocytes (see below) or 1 h incubation in 100 µM diamide in M2.
In vitro fertilization
Spermatozoa released from caudae epididymides of a mature F1 (CBA/HxC57Bl6 or C57Bl6xCBA/H) male mouse were suspended in 0.5 ml fertilization medium and incubated for 1.5 h to allow capacitation and spontaneous acrosome reaction. Sperm concentration was 2x107 spermatozoa/ml. Prior to fertilization, zonae pellucidae were removed by exposure of oocytes to acidic Tyrode's solution (pH 2.5; Nicolson et al. 1975). In some experiments, oocytes were preincubated 30 min with 1, 2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetylester) (BAPTA-AM; 30 µM in M2) before insemination. Oocytes were placed in 100 µl droplets of fertilization medium and 1 µl of the preincubated sperm suspension was added (final concentration of sperm suspension was 2x105 spermatozoa/ml). After 1 h, the oocytes were gently pipetted several times to remove loosely attached spermatozoa and then cultured in M2 for 1–4.5 h. In some experiments insemination, as well as a subsequent culture, were performed in a medium supplemented with cycloheximide (CHX; 10 µg/ml). Moreover, in some experiments fertilized oocytes were stained with a chromatin dye, Hoechst 33342 (100 ng/ml M2), and examined under an inverted fluorescence microscope in order to count the number of spermatozoa inside.
Microinjections and fluorescence and luminescence imaging
Microinjection was carried out as described previously (Saunders et al. 2002). Briefly, maturing or metaphase II oocytes were microinjected using pressure pulses applied to the back of micropipettes inserted into eggs using overcompensation of the negative capacitance of a serially connected electrical amplifier. The volume of solution injected was 3–5% of an oocyte volume. Oocytes were injected with a solution containing 0.5 mM Oregon green BAPTA dextran (OGBD; Invitrogen Ltd) and 0.75 µg/µl cRNA of cyclin B1 tagged with firefly luciferase, (pipette concentrations). Human cyclin B1 from cyclin B1-GFP construct (Nixon et al. 2002) was linked to N-terminus of firefly luciferase (pGL3) and placed into the pCS2+ vector. Polyadenylated cRNA was then generated from the SP6 promoter as described previously (Nixon et al. 2002). Before injection, zonae pellucidae were removed by treatment with acidic Tyrode's solution and then oocytes were attached to a polylysine-coated cover slip that formed the bottom of a heated (37 °C) chamber on an inverted microscope. Oocytes were maintained in HEPES-buffered KSOM medium (potassium simplex optimization medium; Lawitts & Biggers 1993) containing 100 µM luciferin (Dumollard et al. 2004). Oocytes were inseminated by adding 10 µl capacitated sperm suspension. (For these experiments, sperm isolated from two caudae epididymides were incubated in T6 medium for 2 h before use).
Fluorescence (reflecting cytoplasmic concentration of free Ca2+ ions) and luminescence (reflecting cyclin B1-luciferase expression) were both imaged in the same sets of oocytes using a Nikon TE2000 microscope equipped with a 20x0.65NA Fluor objective lens as was described previously (Campbell & Swann 2006). After the measurements, oocytes were stained with Hoechst 33342 (100 ng/ml HEPES-buffered KSOM) and the number of spermatozoa inside the oocytes was counted.
Fusion of oocytes with thymocytes
Maturing oocytes (2–3 h after GVBD) were fused with thymocytes by a polyethylene glycol (PEG) method as described previously (Czolowska et al. 1984). Thymocytes were isolated from thymuses of 1- to 5-day-old mice. Maturing oocytes were exposed to 0.5% solution of pronase in Ringer's saline in order to remove zonae pellucidae and washed in M2. Then oocytes were stained with Hoechst 33342 (100 ng/ml) for 30 min in order to visualize the chromatin. Subsequently, oocytes were washed carefully in M2 without BSA and transferred into phytohemagglutinin (PHA) solution (300 mg/ml in M2 without BSA) for 5 min. Then, they were transferred to M2 without BSA and agglutinated with thymocytes. The oocytes coated with thymocytes were exposed to 40% (w/v) PEG solution (MW2000; Fluka, Poznan, Poland) in BSA-free M2 for 30–45 seconds to induce fusion. Then, the cells were washed in M2 with BSA and cultured in vitro for 1–1.5 h in M2. Dishes containing M2 without BSA, PHA solution, and M2 with BSA were coated with agar (Bacto-Agar, Difco, Lawrence, KS, USA, 1% (w/v) solution in 0.9% (w/v) solution of NaCl). Fusion usually occurred within 15–30 min after exposure to PEG. After 1 h culture in M2, hybrids were examined under a fluorescence microscope in order to calculate the number of thymocyte nuclei inside the oocytes. Since oocytes were incubated in Hoechst 33342 before fusion with thymocytes, only chromatin of thymocytes that fused with oocytes was stained.
Glutathione measurements
To estimate the glutathione concentration in oocytes we used a fluorescent indicator of glutathione, monochlorobimane (MCB; Keelan et al. 2001). Oocytes were incubated with 50 µM MCB in M2 medium for 45 min and then fluorescence of MCB was registered at 390 nm by a digital camera (Pixelfly, PCO, Kelheim, Germany). Intensity of fluorescence was analyzed by Image J software (http://rsb.info.nih.gov/ij/). When glutathione concentration was measured in fertilized oocytes, they were subsequently fixed in 4% paraformaldehyde for 20 min and stained with chromomycin (0.01 mg/ml PBS supplemented with 5 mM MgCl2). Oocytes were then examined under a fluorescence microscope at 450 nm, the chromomycin excitation wavelength, to calculate the number of sperm inside.
Histone H1 kinase assay
The activity of MPF was measured as an activity of histone H1 kinase, according to the method described by Verlhac et al. (1994). Oocytes were washed in PBS and pooled in groups of 5 in 1.5 µl drops of PBS. Samples were frozen and stored in –80 °C until further analysis. Three microliters of a lysing buffer (containing 0.16 M glycerophosphate, 40 mM EGTA (pH 7.3), 30 mM MgCl2, 2 mM dithiothreitol, protease inhibitors (diluted 1:20, Complete Protease Inhibitor Cocktail, Roche), and BSA (11.3 mg/ml); final concentrations) was added to each sample. Oocytes were then lysed by freezing and thawing, and subsequently 1.5 µl reaction buffer (containing 0.5 mg/ml histone H1, 5 mM ATP, and 1.67 µCi/µl [32P]-ATP (MP Biomedicals, Illkirch, France); final concentrations) was added. Samples were incubated for 30 min in 30 °C. The reaction was stopped by an addition of 4 µl Laemmli buffer (Laemmli 1970) per sample. Samples were boiled for 10 min and proceeded for 12% SDS-PAGE. Gels were exposed to autoradiography films (BioMax MS Film, Kodak) at –80 °C for 24–72 h. Intensity of bands on autoradiography films was measured with GelDoc using software Quantity One 4.2.2 (Bio-Rad). The intensity of the bands reflected the intensity of histone H1 phosphorylation and enabled us to quantify histone H1 kinase (MPF) activity.
Immunoblotting
The levels of total CDC2A, CDC2A phosphorylated at Thr 161 and Tyr 15, and 
-tubulin were examined in maturing oocytes that were fertilized in a polyspermic way or treated with diamid. Cell lysates from 180 oocytes were mixed with 4x NuPage LDS sample Buffer and 10x NuPage Sample Reducing Agent (Invitrogen) and were heated for 10 min in 70 °C. The samples were subjected to electrophoresis on NuPage Novex 10% Bis-Tris gels in NuPage MOPS SDS Running Buffer (Invitrogen) and separated proteins were transferred onto PVDF membranes (Hyperbond-P, Amersham Biosciences). PVDF membranes were probed overnight at 4 °C with: 1) a mouse monoclonal antibody (MAB) against total CDC2A (diluted 1:100, Abcam, Cambridge, UK, cat. no. ab18); 2) a rabbit polyclonal antibody anti-CDC2A phosphorylated at pTyr15 (anti-CDC2A-pTyr15, 1:100, Abcam, cat. no. ab10533); 3) a rabbit polyclonal antibody against CDC2A phosphorylated at Thr161 (anti-CDC2A-pThr161, 1:50, Cell Signaling Technology, Danvers, MA, USA, cat. no. 9114); 4) a mouse MAB against -tubulin (1:1000, Sigma–Aldrich, cat. no. T5168). Antibodies against total CDC2A, CDC2A-pTyr15, and -tubulin were diluted in 5% nonfat milk in TBS containing 0.05% Tween 20 (Bio-Rad). Antibody against CDC2A-pThr161 was diluted in 5% BSA in TBS containing 0.1% Tween 20. Subsequently, the membranes were incubated for 1 h with secondary antibodies (goat anti-rabbit or anti-mouse antibodies (Pierce, Rockford, IL, USA) dilution 1:6000) conjugated with HRP. Proteins were detected by the ECL technique using SuperSignal West Dura Extended Duration Substrate reagents (Pierce) according to the manufacturer's instruction.
Cytological examination
For cytological examination maturing oocytes were fixed with Heidenhain's fixative. Whole-mount preparations were stained with hematoxylin according to the method of Tarkowski & Wroblewska (1967).
Statistical analysis
Statistical analysis of the results was performed using the Student's t-test. Results were considered statistically significant when P
0.05.
|
Declaration of interest |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
|---|
|
Funding |
|---|
|
Acknowledgements |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
|---|
Received June 17, 2008
First decision July 28, 2008
Accepted September 10, 2008
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
|---|
Ajduk A & Maleszewski M 2004 Sperm-derived activating ability does not persist in mouse oocytes inseminated during in vitro maturation. Molecular Reproduction and Development 68 240–249.[Medline]
Ajduk A, Malagocki A & Maleszewski M 2008 Cytoplasmic maturation of mammalian oocytes: development of a mechanism responsible for sperm-induced Ca2+ oscillations. Reproductive Biology 8 3–22.[Medline]
Balhorn R 1982 A model for the structure of chromatin in mammalian sperm. Journal of Cell Biology 93 298–305.
Brandeis M, Rosewell I, Carrington M, Crompton T, Jacobs MA, Kirk J, Gannon J & Hunt T 1998 Cyclin B2-null mice develop normally and are fertile whereas cyclin B1-null mice die in utero. PNAS 95 4344–4349.
Brunet S, Maria AS, Guillaud P, Dujardin D, Kubiak JZ & Maro B 1999 Kinetochore fibers are not involved in the formation of the first meiotic spindle in mouse oocytes, but control the exit from the first meiotic M phase. Journal of Cell Biology 146 1–12.[CrossRef][Web of Science][Medline]
Campbell K & Swann K 2006 Ca2+ oscillations stimulate an ATP increase during fertilization of mouse eggs. Developmental Biology 298 225–233.[CrossRef][Medline]
Carroll J, Swann K, Whittingham D & Whitaker M 1994 Spatiotemporal dynamics of intracellular [Ca2+]i oscillations during the growth and meiotic maturation of mouse oocytes. Development 120 3507–3517.[Abstract]
Cheng A, Ross KE, Kaldis P & Solomon MJ 1999 Dephosphorylation of cyclin-dependent kinases by type 2C protein phosphatases. Genes and Development 13 2946–2957.
Chesnel F, Vignaux F, Richard-Parpaillon L, Huguet A & Kubiak JZ 2005 Differences in regulation of the first two M-phases in Xenopus laevis embryo cell-free extracts. Developmental Biology 285 358–375.[CrossRef][Medline]
Chesnel F, Bazile F, Pascal A & Kubiak JZ 2006 Cyclin B dissociation from CDK1 precedes its degradation upon MPF inactivation in mitotic extracts of Xenopus laevis embryos. Cell Cycle 5 1687–1698.[Web of Science][Medline]
Chesnel F, Bazile F, Pascal A & Kubiak JZ 2007 Cyclin B2/cyclin-dependent kinase1 dissociation precedes CDK1 Thr-161 dephosphorylation upon M-phase promoting factor inactivation in Xenopus laevis cell-free extract. International Journal of Developmental Biology 51 297–305.[CrossRef][Web of Science][Medline]
Cheung A, Swann K & Carroll J 2000 The ability to generate normal Ca2+ transients in response to spermatozoa develops during the final stages of oocyte growth and maturation. Human Reproduction 15 1389–1395.
Ciemerych MA & Kubiak JZ 1998 Cytostatic activity develops during meiosis I in oocytes of LT/Sv mice. Developmental Biology 200 198–211.[CrossRef][Web of Science][Medline]
Clarke HJ & Masui Y 1983 The induction of reversible and irreversible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes. Developmental Biology 97 291–301.[CrossRef][Web of Science][Medline]
Clarke HJ & Masui Y 1986 Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse. Journal of Cell Biology 102 1039–1046.
Clarke HJ & Masui Y 1987 Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes. Journal of Cell Biology 104 831–840.
Czolowska R, Modlinski JA & Tarkowski AK 1984 Behaviour of thymocyte nuclei in non-activated and activated mouse oocytes. Journal of Cell Science 69 19–34.[Abstract]
D'Angiolella V, Palazzo L, Santarpia C, Costanzo V & Grieco D 2007 Role for non-proteolytic control of M-phase promoting factor activity at M-phase exit. PLoS ONE 2 e247.[CrossRef]
Draviam VM, Orrechia S, Lowe M, Pardi R & Pines J 2001 The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and neither enzyme requires MEK to disassemble the Golgi apparatus. Journal of Cell Biology 152 945–958.
Dumollard R, Marangos P, Fitzharris G, Swann K, Duchen M & Carroll J 2004 Sperm-triggered Ca2+ oscillations and Ca2+ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production. Development 131 3057–3067.
Dumollard R, Ward Z, Carroll J & Duchen MR 2007 Regulation of redox metabolism in the mouse oocyte and embryo. Development 134 455–465.
Eppig JJ, Schultz RM, O'Brien M & Chesnel F 1994 Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes. Developmental Biology 164 1–9.[CrossRef][Web of Science][Medline]
Fang G 2002 Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex. Molecular Biology of the Cell 13 755–766.
Fraser L 1982 Ca2+ is required for mouse sperm capacitation and fertilization in vitro. Journal of Andrology 3 412–419.[Abstract]
Fulton BP & Whittingham DG 1978 Activation of mammalian oocytes by intracellular injection of calcium. Nature 273 149–151.
Garrett S, Barton WA, Knights R, Jin P, Morgan DO & Fisher RP 2001 Reciprocal activation by cyclin-dependent kinases 2 and 7 is directed by substrate specificity determinants outside the T loop. Molecular and Cellular Biology 21 88–99.
Gorr IH, Reis A, Boos D, Wühr M, Madgwick S, Jones KT & Stemmann O 2006 Essential CDK1-inhibitory role for separase during meiosis I in vertebrate oocytes. Nature Cell Biology 8 1035–1037.[CrossRef][Web of Science][Medline]
Hansen DV, Tung JJ & Jackson PK 2006 CaMKII and polo-like kinase 1 sequentially phosphorylate the cytostatic factor Emi2/XErp1 to trigger its destruction and meiotic exit. PNAS 103 608–613.
Hoffmann I, Clarke PR, Marcote MJ, Karsenti E & Draetta G 1993 Phosphorylation and activation of human cdc25-C by cdc2–cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO Journal 12 53–63.[Web of Science][Medline]
Homer HA, McDougall A, Levasseur M, Murdoch AP & Herbert M 2005 Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes. Reproduction 130 829–843.
Hyslop LA, Nixon VL, Levasseur M, Chapman F, Chiba K, McDougall A, Venables JP, Elliott DJ & Jones KT 2004 Ca2+-promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest. Developmental Biology 269 206–219.[CrossRef][Web of Science][Medline]
Jackman M, Firth M & Pines J 1995 Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the Golgi apparatus. EMBO Journal 14 1646–1654.[Medline]
Jones KT, Carroll J & Whittingham DG 1995 Ionomycin, thapsigargin, ryanodine, and sperm induced Ca2+ release increase during meiotic maturation of mouse oocytes. Journal of Biological Chemistry 270 6671–6677.
Josefsberg LB, Kaufman O, Galiani D, Kovo M & Dekel N 2001 Inactivation of M-phase promoting factor at exit from first embryonic mitosis in the rat is independent of cyclin B1 degradation. Biology of Reproduction 64 871–878.
Kaushal N & Bansal MP 2007 Inhibition of CDC2/Cyclin B1 in response to selenium-induced oxidative stress during spermatogenesis: potential role of Cdc25c and p21. Molecular and Cellular Biochemistry 298 139–150.[Medline]
Keelan J, Allen NJ, Antcliffe D, Pal S & Duchen MR 2001 Quantitative imaging of glutathione in hippocampal neurons and glia in culture using monochlorobimane. Journal of Neuroscience Research 66 873–884.[CrossRef][Web of Science][Medline]
Krek W & Nigg EA 1991 Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates. EMBO Journal 10 3331–3341.[Web of Science][Medline]
Kubiak JZ 1989 Mouse oocytes gradually develop the capacity for activation during the metaphase II arrest. Developmental Biology 136 537–545.[CrossRef][Web of Science][Medline]
Kubiak JZ, Weber M, Géraud G & Maro B 1992 Cell cycle modification during the transitions between meiotic M-phases in mouse oocytes. Journal of Cell Science 102 457–467.
Kubiak JZ, Weber M, de Pennart H, Winston NJ & Maro B 1993 The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF. EMBO Journal 12 3773–3778.[Web of Science][Medline]
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 680–685.[CrossRef][Web of Science][Medline]
Lawitts JA & Biggers JD 1993 Culture of preimplantation embryos. Methods in Enzymology 225 153–164.[Web of Science][Medline]
Ledan E, Polanski Z, Terret ME & Maro B 2001 Meiotic maturation of the mouse oocyte requires an equilibrium between cyclin B synthesis and degradation. Developmental Biology 232 400–413.[CrossRef][Web of Science][Medline]
Lorca T, Labbe JC, Devault A, Fesquet D, Capony JP, Cavadore JC, Le Bouffant F & Doree M 1992 Dephosphorylation of cdc2 on threonine 161 is required for cdc2 kinase inactivation and normal anaphase. EMBO Journal 11 2381–2390.[Web of Science][Medline]
Luberda Z 2005 The role of glutathione in mammalian gametes. Reproductive Biology 5 5–17.[Medline]
Madgwick S, Hansen DV, Levasseur M, Jackson PK & Jones KT 2006 Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis. Journal of Cell Biology 174 791–801.
Maluchnik D & Borsuk E 1994 Sperm entry into fertilised mouse eggs. Zygote 2 129–131.[Medline]
Marangos P & Carroll J 2004 Fertilization and InsP3-induced Ca2+ release stimulate a persistent increase in the rate of degradation of cyclin B1 specifically in mature mouse oocytes. Developmental Biology 272 26–38.[CrossRef][Web of Science][Medline]
Margalit A, Vlcek S, Gruenbaum Y & Foisner R 2005 Breaking and making of the nuclear envelope. Journal of Cellular Biochemistry 95 454–465.[CrossRef][Medline]
Martinez AM, Afshar M, Martin F, Cavadore JC, Labbe JC & Doree M 1997 Dual phosphorylation of the T-loop in cdk7: its role in controlling cyclin H binding and CAK activity. EMBO Journal 16 343–354.[CrossRef][Web of Science][Medline]
McLay DW & Clarke HJ 2003 Remodelling the paternal chromatin at fertilization in mammals. Reproduction 125 625–633.[Abstract]
Mueller PR, Coleman TR & Dunphy WG 1995 Cell cycle regulation of a Xenopus Wee1-like kinase. Molecular Biology of the Cell 6 119–134.[Abstract]
Murray AW, Solomon MJ & Kirschner MW 1989 The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature 339 280–286.[CrossRef][Medline]
Nicolson GL, Yanagimachi R & Yanagimachi H 1975 Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs. Journal of Cell Biology 66 263–274.
Nishiyama A, Tachibana K, Igarashi Y, Yasuda H, Tanahashi N, Tanaka K, Ohsumi K & Kishimoto T 2000 A nonproteolytic function of the proteasome is required for the dissociation of Cdc2 and cyclin B at the end of M phase. Genes and Development 14 2344–2357.
Nixon VL, Levasseur M, McDougall A & Jones KT 2002 Ca2+ oscillations promote APC/C-dependent cyclin B1 degradation during metaphase arrest and completion of meiosis in fertilizing mouse eggs. Current Biology 12 746–750.[CrossRef][Web of Science][Medline]
Parker LL & Piwnica-Worms H 1992 Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. Science 257 1955–1957.
Perreault SD, Wolff RA & Zirkin BR 1984 The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo. Developmental Biology 101 160–167.[CrossRef][Web of Science][Medline]
Perreault SD, Barbee RR & Slott VL 1988 Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Developmental Biology 125 181–186.[CrossRef][Web of Science][Medline]
Poon RY, Yamashita K, Adamczewski JP, Hunt T & Shuttleworth J 1993 The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2. EMBO Journal 12 3123–3132.[Web of Science][Medline]
Pyrzynska B, Maleszewski M & Maluchnik D 1996 Mouse oocytes penetrated by sperm at GV or GVBD stage lose the ability to fuse with additional spermatozoa. Zygote 4 123–128.[Web of Science][Medline]
Quinn P, Whittingham DG & Stanger JD 1982 Interaction of semen with ova in vitro. Archives of Andrology 8 189–198.[Medline]
Rauh NR, Schmidt A, Bormann J, Nigg EA & Mayer TU 2005 Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation. Nature 437 1048–1052.[CrossRef][Medline]
Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann K & Lai FA 2002 PLC zeta: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development. Development 129 3533–3544.
Savitsky PA & Finkel T 2002 Redox regulation of Cdc25C. Journal of Biological Chemistry 277 20535–20540.
Shoji S, Yoshida N, Amanai M, Ohgishi M, Fukui T, Fujimoto S, Nakano Y, Kajikawa E & Perry AC 2006 Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20. EMBO Journal 25 834–845.[CrossRef][Web of Science][Medline]
De Smedt V, Poulhe R, Cayla X, Dessauge F, Karaiskou A, Jessus C & Ozon R 2002 Thr-161 phosphorylation of monomeric Cdc2. Regulation by protein phosphatase 2C in Xenopus oocytes. Journal of Biological Chemistry 277 28592–28600.
Sohn J & Rudolph J 2003 Catalytic and chemical competence of regulation of cdc25 phosphatase by oxidation/reduction. Biochemistry 42 10060–10070.[CrossRef][Medline]
Solomon MJ, Lee T & Kirschner MW 1992 Role of phosphorylation in p34cdc2 activation: identification of an activating kinase. Molecular Biology of the Cell 3 13–27.[Abstract]
Strausfeld U, Labbe JC, Fesquet D, Cavadore JC, Picard A, Sadhu K, Russell P & Doree M 1991 Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein. Nature 351 242–245.[CrossRef][Medline]
Sudakin V, Chan GK & Yen TJ 2001 Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. Journal of Cell Biology 154 925–936.
Tang Z, Bharadwaj R, Li B & Yu H 2001 Mad2-Independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1. Developmental Cell 1 227–237.[CrossRef][Web of Science][Medline]
Tarkowski AK & Wroblewska J 1967 Development of blastomeres of mouse eggs isolated at the 4- and 8-cell stage. Journal of Embryology and Experimental Morphology 18 155–180.[Web of Science][Medline]
Tsurumi C, Hoffmann S, Geley S, Graeser R & Polanski Z 2004 The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes. Journal of Cell Biology 167 1037–1050.
Tung JJ, Hansen DV, Ban KH, Loktev AV, Summers MK, Adler JR III & Jackson PK 2005 A role for the anaphase-promoting complex inhibitor Emi2/XErp1, a homolog of early mitotic inhibitor 1, in cytostatic factor arrest of Xenopus eggs. PNAS 102 4318–4323.
Verlhac MH, Kubiak JZ, Clarke HJ & Maro B 1994 Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes. Development 120 1017–1025.[Abstract]
Winston NJ 1997 Stability of cyclin B protein during meiotic maturation and the first mitotic cell division in mouse oocytes. Biology of the Cell 89 211–219.[CrossRef][Web of Science][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
