| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gamete Biology |
Department of Applied Genetics,3 Graduate School of Agriculture and Life Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
Division of Biological Sciences,4 Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-0810, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
gamete biology, kinases, meiosis, oocyte development, ovum
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In addition to Xenopus oocytes, immature oocytes of marine invertebrates, such as starfish and surf clam, contain abundant cyclin B as a component of pre-MPF, a hyperphosphorylated inactive MPF [10, 11]. These oocytes do not require cyclin B synthesis for GVBD induction, because the MPF is activated by the dephosphorylation of pre-MPF. The presence of cyclin B in immature oocytes and the dispensability of its synthesis for GVBD induction has also been reported in mouse oocytes [12–14]. On the other hand, immature oocytes in goldfish and R. japonica have no cyclin B, and their meiotic resumption completely depends on cyclin B synthesis [15–17]. It has been known that in many mammalian oocytes apart from those of rodents, some kind of protein synthesis is required for GVBD induction; however, the specific protein has not yet been identified [18]. The absence of CB1 in immature bovine oocytes [19, 20] and the presence of only an extremely small amount of pre-MPF in immature porcine oocytes [8] suggest the possibility that cyclin B is the protein whose synthesis is required for GVBD induction.
In the present study, we first examined the protein levels of CB1 and CB2 simultaneously throughout porcine oocyte maturation; then their roles in maturation were analyzed by separately inhibiting the synthesis of each with its specific antisense RNA. Finally, we inhibited both of their syntheses by injecting a mixture of their antisense RNAs. We discuss the requirement of cyclin B synthesis for GVBD induction in porcine oocytes.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ovaries of prepubertal gilts were collected at a commercial slaughterhouse and transported to the laboratory at about 37°C in saline. Cumulus-oocyte complexes (COCs) were aspirated from follicles (about 2–5 mm in diameter) and washed four times in a modified Krebs-Ringer bicarbonate solution [21] containing 20% porcine follicular fluid, 1.0 IU/ml eCG (Pramex; Sankyo, Tokyo, Japan), and 3.2 mg/ml BSA (fraction V; WAKO Pure Chemical Ind., Osaka, Japan). The washed COCs were subjected to microinjection as described below. Groups of 10–20 COCs were cultured for up to 48 h in 100 µl of this medium, covered by liquid paraffin (Nakalai Tesque Inc., Kyoto, Japan) at 37°C, 5% CO2 in air and saturated humidity. After culturing, the surrounding cumulus cells were removed by treatment with 150 IU/ml hyaluronidase (type IV, Sigma, St. Louis, MO) and gentle pipetting in saline supplemented with 0.1% polyvinyl-pyrrolidone (PVP, average molecular weight 10 000; Sigma). The denuded oocytes were subjected to immunoblotting and MPF activity assay. Some oocytes were examined for nuclear status after being mounted on a gross slide, fixed with acetic acid-ethanol (1:3), and stained with 0.75% aceto-orcein solution.
Preparation of Porcine Cyclin B1 and Cyclin B2 Antisense RNAs
Fragments of porcine CB1 and CB2 cDNA were obtained by RT-PCR of total RNA extracted from porcine noncultured oocytes using a commercial RNA extraction solution (Trizo Reagent; Gibco BRL, Karlsruhe, Germany). The primer pairs were designed according to the sequences of porcine CB1 and human CB2 as follows: CB1 forward primer, 5'-AA-GAGCATTAAATTTTGGTCTGGG-3'; CB1 reverse primer, 5'-CTTTGT-AAGCCCTCGATTCACCACG-3'; CB2 forward primer, 5'- AA-AGTTGGCTCCAAAGGGTCCTT-3'; and CB2 reverse primer, 5'- GAAACTGGCTGAACCTGTAAAAAT-3'. The primer positions and expected product lengths are shown in Figure 2A. The PCR products derived from porcine CB1 primer pairs and human CB2 primer pairs were cloned into pGEM-T Easy vector (A1360, Promega, Madison, WI); the resulting vectors are referred to as B1/pGEM-T and B2/pGEM-T, respectively. The PCR product inserted into B2/pGEM-T was sequenced using a commercial sequencing kit (Thermo Sequence Cycle Sequencing Kit, P/N 78500, USB Corp., Cleveland, OH) and a DNA sequencer (ALF Express, Amersham-Pharmacia Biotech, Tokyo, Japan).
|
For the in vitro synthesis of antisense RNAs, B1/pGEM-T and B2/pGEM-T were linearized by cutting with Sal I (Takara Shuzo Co., Ltd., Tokyo, Japan); the linearized cDNA then was transcribed in vitro with T7-mRNA-polymerase using the Cap-Scribe system (Nippon Roche Co., Ltd., Kamakura, Japan) according to the manufacturer's instructions. The reaction was performed in the presence of m7G(5')ppp(5')G to synthesize capped RNA transcripts. The enhanced green fluorescent protein (EGFP) mRNA was also prepared from EGFP/pGEM-3Z and the same in vitro transcription system as described previously [22]. The RNA transcripts were precipitated with absolute ethanol, washed, dried, and resuspended in RNase-free water. The RNA solutions were stored at -80°C until use.
Microinjection
In a previous report, we found that co-injecting EGFP mRNA with other mRNAs or antisense RNAs and then collecting the oocytes with EGFP illumination was a powerful method for selecting the viable oocytes [22, 23]. Therefore, we employed this method for the injection of CB1 and/or CB2 antisense RNAs as a marker of oocyte viability. About 30% of oocytes were EGFP-positive. The concentration of each RNA in the solutions was adjusted to 0.5 µg/µl. The microinjection was performed using microinjectors (IM-5A/B; Narisige, Tokyo, Japan) equipped with manipulators (Motorsteuerung; Zeiss, Oberkochen, Germany) mounted on an inverted microscope (Zeiss). Approximately 50 pl of RNA solution was injected into each ooplasm by continuous pneumatic pressure using a holding pipette (outer diameter 150 µm, inner diameter 50 µm) and an injection pipette (diameter less than 0.1 µm) sterilized by heating (200°C, 2 h). After injection, all COCs were cultured as described above and the expression of EGFP was examined under a fluorescent stereomicroscope (MZ FLIII, Leica, Wetzlar, Germany). Only the oocytes expressing EGFP illumination were used for analyses in the present study.
MPF Activity Assay
Ten denuded oocytes were lysed in 2.5 µl assay buffer [24] and stored at -80°C until use. The activity of MPF was evaluated using the histone H1 kinase activity as described in previous reports [24]. The lysates (2.5 µl) were added to 2.5 µl of cAMP-dependent protein kinase inhibitor (2.5 µM; Sigma), 5 µl of histone H1 (2 mg/ml; Sigma), and 5 µl of [
-32P]ATP (0.1 mM, 0.4 mCi/ml; Amersham Pharmacia Biotech, Buckinghamshire, England), and the reaction was incubated at 37°C for 1 h. Next, 5 µl of 5x Laemmli buffer [25] was added to each lysate, which was then denatured at 100°C for 5 min and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The bands of phosphorylated histone H1 were visualized after autoradiography.
Immunoblotting
Micro-Western blotting method [26] was used with several modifications. In all cases except for that in Figure 1B, 10 oocytes were put in 2 µl of saline supplemented with 0.1% PVP, added 0.5 µl of 5x Laemmli buffer, and denatured at 100°C for 5 min. In Figure 1B, the indicated numbers of oocytes were subjected to the same treatment. Proteins were separated on a 10% polyacrylamide gel by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (AE-6660; Atto Co., Tokyo, Japan). After blocking the membrane with 5% milk for 1 h, the membrane was treated with anti-CB1 monoclonal antibody (CB169, Upstate Biochemistry Inc., Waltham, MA); anti-CB2 polyclonal antibody (N-20, Santa Cruz Biotechnology, Santa Cruz, CA); or anti-cdc2 monoclonal antibody [27]. Signals were detected by an ECL blotting detection kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
|
Statistical Analysis
Chi square test and Student t-test were used for evaluation of the results. Probability of P < 0.05 was considered to be statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Figure 1 shows protein levels of CB1, CB2, and cdc2 during maturation of porcine oocytes (Fig. 1, A and B) with MPF activity (Fig. 1C) and meiotic states (Fig. 1D). A faint 45 kDa band of CB2 was first detected at 18 h of culture (also see Fig. 4A), when each of 10 oocytes was used for immunoblotting; this correlated with an upward trend of MPF activity (Fig. 1C). CB1 was detected at 62 kDa after 24 h of culture when almost all oocytes had undergone GVBD (Fig. 1D) and the MPF activity had reached its maximum level (Fig. 1C). Thereafter, CB1 gradually increased with a transient decrease at 36 h and reached its peak level at 48 h of culture. The CB2 level was higher than that of CB1 at 24 and 30 h of culture, correlating with the first meiotic metaphase (Fig. 1D), then decreased drastically after 36 h of culture and remained at a low level until 48 h. Almost no change was detected in the cdc2 level during maturation of oocytes. The appearance of a shift-up band at 40 and 48 h has been explained previously as the accumulation of pre-MPF [8].
|
As both CB1 and CB2 were undetectable in noncultured oocytes when only 10 oocytes were used for immunoblotting, we examined the amounts of these cyclins in detail using pools of 200 noncultured oocytes and compared them with pools of 0, 5, 10, 20, and 40 oocytes that had been cultured for 24 h (first metaphase). As shown in Figure 1B, the intensity of the CB2 band was similar to that observed from the 5 and 10 oocyte pools of 24-h cultured oocytes, showing that the CB2 amount in noncultured oocytes was between 1/20 and 1/40 of that in 24-h cultured oocytes. No band was observed for CB1.
Partial Sequence of Porcine Cyclin B2
As the sequence of porcine CB2 was not available, PCR primers were designed according to human CB2 at the positions shown in Figure 2A. By using these primer pairs, an RT-PCR product was obtained at the expected base length (about 360 bases) from total RNA extracted from noncultured porcine oocytes. The PCR product was isolated and its sequence was examined; the sequence is shown with the corresponding part of human CB2 in Figure 2B. The porcine PCR product had 354 bases, 88% of which were identical to the human CB2.
Effects of Cyclin B1 Antisense RNA Injection on Meiotic Maturation of Porcine Oocytes
The injection of CB1 antisense RNA into cytoplasm of porcine oocytes at 0 h of culture completely inhibited CB1 synthesis throughout the maturation period without any effects on the CB2 level (Fig. 3A). Although the MPF activity of injected oocytes had increased normally at 18 h of culture, the activity did not elevate thereafter and did not reach the maximum level until 48 h of culture. The activity of CB1-deficient oocytes was significantly lower than that of control oocytes after 24 h of culture (Fig. 3B). In spite of the low MPF activity in the injected oocytes, GVBD occurred at the normal rate and over the normal time course, reaching the first meiotic metaphase normally as shown in Figure 3C. The oocytes were morphologically normal during first meiosis and extruded first polar bodies normally; however, the absence of CB1 had a drastic impact on the second meiotic arrest of the matured oocytes (Fig. 3C). Almost all oocytes formed a pronucleus at 48 h of culture (data not shown).
|
Effects of Cyclin B2 Antisense RNA Injection on Meiotic Maturation of Porcine Oocytes
The injection of CB2 antisense RNA into cytoplasm of porcine oocytes at 0 h of culture inhibited CB2 synthesis throughout the maturation period without any effects on the CB1 level (Fig. 4A). Although the MPF activity at 18 h of culture tended to be lower in injected oocytes than that in control oocytes, the activity rose to the maximum level at 24 h of culture, and thereafter the normal high activity was maintained until 48 h of culture. The activity of the injected oocytes was not significantly different than that of control oocytes throughout the maturation period (Fig. 4B). The absence of CB2 synthesis had almost no effect on meiotic progression including GVBD induction, morphology of the first meiotic metaphase, and arrest at the second meiotic metaphase (Fig. 4C). The only effect was the short duration of the first meiotic metaphase and accelerated extrusion of the first polar body (Fig. 4C).
Effects of Cyclin B1 and Cyclin B2 Antisense RNA Mixture Injection on Meiotic Maturation of Porcine Oocytes
The injection of the mixture of CB1 and CB2 antisense RNAs into cytoplasm of porcine oocytes at 0 h of culture completely inhibited the synthesis of both cyclins throughout the maturation period (Fig. 5A). The MPF activity of injected oocytes was low throughout the maturation period, although the activity gradually increased with the culture period (Fig. 5B). At 24 h of culture, the GVBD rate in the cyclin B-inhibited oocytes was dramatically low (15.1%) compared with that in control oocytes (70.2%), but the rate gradually increased thereafter (37.0% and 62.8% at 30 h and 48 h, respectively), as shown in Figure 5C. About half of the oocytes that had undergone GVBD formed chromosome clusters like prometaphase, but the other half were morphologically normal until the first polar body extrusion and formed pronuclei, similar to CB1-deficient oocytes (data not shown).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The increase of CB2 was observed just before GVBD and reached its peak level at the first metaphase, then decreased abruptly, correlating with the first polar body extrusion, and maintained a low level during the second meiosis. In contrast, CB1 was first detected at 24 h when most oocytes underwent GVBD, as reported previously [8], and increased gradually until 48 h of culture. In Xenopus oocytes, the degradation of CB2 at the first meiosis (MI)/second meiosis (MII) transition and the requirement of CB1 synthesis for the MII induction have been reported [13]. Except for the cyclin B levels in immature oocytes, the present cyclin B fluctuation patterns agreed well with those reported in Xenopus and mouse oocytes [3, 9, 30]. These reports and the present results prompt us to conclude that CB2 works during MI, whereas CB1 works mainly during MII. The oocytes injected with CB1 antisense RNA underwent MI in a normal time course and at normal rates, whereas their subsequent MII became abnormal and almost no oocytes stopped at the second meiotic metaphase but continued to form a pronucleus. This result agrees well with our conclusion. The low MPF activity in those oocytes indicates the requirement of high MPF activity induced by CB1 synthesis for maintaining the second meiotic arrest. In mouse oocytes, the formation of a pronucleus was reported when CB1, but not CB2, synthesis was inhibited [14], indicating that the second meiosis is regulated mainly by CB1 in mouse oocytes as well. Taken together, the mouse study and our porcine study suggest the consistency of cyclin B roles in mammals.
As porcine CB2 has not yet been isolated and its sequence was not available, we tried partial cloning of porcine CB2 based on the human CB2 sequence and obtained a fragment that was 88% homologous with the corresponding region of human CB2 [31]. The specific inhibition of CB2 synthesis in porcine oocytes injected with the antisense RNA of this fragment strongly supports the certainty of the present fragment and shows the high conservation of the cyclin B sequence between humans and pigs. To our surprise, GVBD induction in the oocytes injected with CB2 antisense RNA was almost normal, in spite of the expected importance of CB2 in MI. Furthermore, obvious morphological abnormality was not detected in the first meiotic metaphase and first polar body extrusion, although CB2 has been shown to be critical for the first meiotic spindle formation in frog oocytes [4, 5]. The only detected effect of CB2 suppression was the acceleration of first polar body extrusion, suggesting the maintenance effect of CB2 for the first metaphase. It has been reported that CB2-deficient mice were normal, in stark contrast to the morbidity of CB1-deficient mice [32]. The wide rages of CB1 roles and the restricted roles of CB2 also have been reported in cultured human cells [7]. Therefore, it should be considered that most of the roles of CB2 could be compensated for by CB1 in porcine oocytes. These reports and the present study suggest that CB1 is the principal molecule for M-phase regulation in mammalian cells and that CB2 has only an accessory role.
It has been known that some kind of protein synthesis is required for GVBD induction in porcine oocytes, and cyclin B has been considered as one of the candidate proteins whose synthesis is required for GVBD induction. It should be noted that the oocytes injected with both CB1 and CB2 antisense RNAs underwent GVBD gradually, although cyclin B synthesis was not detected in them. Their MPF activity was significantly lower than that in control oocytes but tended to increase gradually. This result shows that the small amount of pre-MPF present in the immature oocytes was activated without de novo cyclin B synthesis and could induce GVBD, although the time course was clearly slow. It could be concluded that in porcine oocytes cyclin B synthesis was not necessary for GVBD induction per se, but synthesis of one cyclin B, either CB1 or CB2, was necessary for GVBD induction in a normal time course. This situation is the intermediate between those oocytes whose GVBD was not affected by the inhibition of cyclin B synthesis, such as Xenopus and mouse [12–14], and those oocytes whose GVBD was completely prevented by the inhibition of cyclin B synthesis, such as R. japonica [17]. This result agrees well with the cyclin B amount in the immature oocytes described above. The present result rules out cyclin B as the candidate protein, although the dispensability of Mos and cdc25 synthesis for GVBD has already been shown in porcine oocytes [23, 33]. Recently, RINGO and Speedy have been isolated separately from Xenopus oocytes [34, 35], and has been shown to induce GVBD in Xenopus as well as mouse oocytes [36]. In preliminary experiments, we have succeeded in partial cloning of porcine RINGO cDNA by RT-PCR from total RNA extracted from immature porcine oocytes (data not shown). Therefore, RINGO should be one of the candidates for the protein whose synthesis is required for GVBD induction in porcine oocytes. In bovine immature oocytes, the absence of CB1 [19, 20] and the presence of CB2 [37] have been reported. As these situations and the requirement of protein synthesis for GVBD induction were the same between bovine and porcine oocytes, it should be assumed that this type of GVBD induction is general in many mammalian oocytes other than rodents.
|
FOOTNOTES |
|---|
2 Correspondence: Kunihiko Naito, Department of Applied Genetics, Graduate School of Agriculture and Life Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. FAX: 81 3 5841 8191; aknaito{at}mail.ecc.u-tokyo.ac.jp
Received: 30 July 2003.
First decision: 18 August 2003.
Accepted: 2 September 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  T. Yamamuro, K. Kano, and K. Naito Functions of FZR1 and CDC20, Activators of the Anaphase-Promoting Complex, During Meiotic Maturation of Swine Oocytes Biol Reprod, December 1, 2008; 79(6): 1202 - 1209. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Endo, K. Kano, and K. Naito Nuclear Histone Deacetylases Are Not Required for Global Histone Deacetylation During Meiotic Maturation in Porcine Oocytes Biol Reprod, June 1, 2008; 78(6): 1073 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Mayes, M. F. Laforest, C. Guillemette, R. B. Gilchrist, and F. J. Richard Adenosine 5'-Monophosphate Kinase-Activated Protein Kinase (PRKA) Activators Delay Meiotic Resumption in Porcine Oocytes Biol Reprod, April 1, 2007; 76(4): 589 - 597. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kume, T. Endo, Y. Nishimura, K. Kano, and K. Naito Porcine SPDYA2 (RINGO A2) Stimulates CDC2 Activity and Accelerates Meiotic Maturation of Porcine Oocytes Biol Reprod, March 1, 2007; 76(3): 440 - 447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Wu, Q. C.-K. Cheung, L. Wen, and J. Li A Growth-Maturation System That Enhances the Meiotic and Developmental Competence of Porcine Oocytes Isolated from Small Follicles Biol Reprod, October 1, 2006; 75(4): 547 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Endo, K. Naito, S. Kume, Y. Nishimura, K. Kashima, and H. Tojo Activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are not required for the global histone deacetylation observed after germinal vesicle breakdown (GVBD) in porcine oocytes. Reproduction, March 1, 2006; 131(3): 439 - 447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Sugiura, K. Naito, T. Endo, and H. Tojo Study of Germinal Vesicle Requirement for the Normal Kinetics of Maturation/M-Phase-Promoting Factor Activity During Porcine Oocyte Maturation Biol Reprod, March 1, 2006; 74(3): 593 - 600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Craig, H. Zhu, P. W. Dyce, J. Petrik, and J. Li Leptin Enhances Oocyte Nuclear and Cytoplasmic Maturation via the Mitogen-Activated Protein Kinase Pathway Endocrinology, November 1, 2004; 145(11): 5355 - 5363. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
