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Regular Article |
a Department of Veterinary Pathobiology
b Department of Animal Science, University of Missouri-Columbia, Columbia, Missouri 65211
c State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, gamete biology, in vitro fertilization, kinases, meiosis, oocyte development, ovum, phosphatases, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Maturation promoting factor (MPF), a complex formed by cyclin B and Cdc2 kinase, is a pivotal regulator of meiosis reinitiation (for a review see [1]).
Mitogen-activated protein (MAP) kinase, a serine/threonine kinase, is another principal regulator of oocyte maturation, and its action in regulating cell cycle events may be uncoupled from MPF in mammalian oocytes (for a review see [2]). MAP kinase is activated by MAP kinase kinase (MAPKK or MEK), which in turn, is activated by Mos protein in the cascade. We and other researchers have shown that MAP kinase is activated after GVBD in mouse [3–5] and rat oocytes [6–8]; however, on the other hand, inhibition of meiosis resumption by cAMP or protein kinase C (PKC) stimulators occurs simultaneously with the inhibition of MAP kinase [5, 7, 9]. Our studies also show that the ability of oocyte cytoplasm to phosphorylate MAP kinase is a prerequisite for the occurrence of GVBD [5, 7]. Furthermore, activation of MAP kinase induces precocious GVBD in meiotic-incompetent oocytes in rodents [10, 11]. Thus, MAP kinase activation is not required for GVBD, but artificial activation of this kinase induces GVBD in rodent oocytes.
Protein phosphatases 1
and 2A have been recently identified in mouse oocytes [12, 13]. Recent studies by us and others show that, in rodent oocytes, protein phosphatases control MAP kinase activation and microtubule organization [6], and inhibition of protein phosphatases not only induces meiosis resumption [14] but also overcomes the inhibitory effects of cAMP or PKC activation on meiosis resumption [5, 7]. Spindle organization is also affected by protein phosphatase inhibitor okadaic acid (OA) in MII-arrested mouse oocytes [15, 16]. However, the temporal relationship among various biochemical and morphological events caused by protein phosphatase inhibition has not been investigated in oocytes of any mammalian species.
Pig oocyte maturation may be regulated by different mechanisms than those of mouse oocytes [17], and they provide better material for studying the phosphatase regulation of MAP kinase phosphorylation, chromatin behavior, microtubule organization, or cell cycle progression during oocyte maturation, because it takes longer for pig oocytes to go through GVBD. In pig and cattle, treatment of oocytes with OA results in acceleration of GVBD and of H1 kinase activity. However, although OA is able to completely release the inhibitory effects exerted by cycloheximide on H1 kinase activity, GVBD occurs in only two thirds of pig oocytes and one quarter of cattle oocytes, even after 20 h of culture [18]. Thus, MPF activation is not sufficient for inducing GVBD under these experimental conditions.
We recently reported that incompetent pig oocyte cytoplasm failed to phosphorylate MAP kinase after 44 h of culture [19]. Another recent report, on the other hand, indicated that microinjection of active MAP kinase into pig oocyte germinal vesicle markedly accelerated GVBD [20]. However, the same authors showed that significant MAP kinase activation was only detected in maturing MI stage oocytes [19, 21]. Taking into consideration all of the published results in pig oocytes, we find that the relationship between MAP kinase phosphorylation and GVBD is still unclear [17–22]. In different maturation systems, in which the meiotic maturation time course differs, MAP kinase activity is at a low level when most of the oocytes go through or go beyond GVBD, and MAP kinase is fully phosphorylated when most oocytes reach or go beyond MI stage [17, 21, 22]. It appears that pig oocyte GVBD may occur in the absence of MAP kinase phosphorylation, or when MAP kinase phosphorylation is at a very low level. Furthermore, GVBD can occur in the absence of MAP kinase phosphorylation in some experimental conditions [23]. Recent studies that we and others have performed show that active MAP kinase is associated with the meiotic spindle after GVBD in pig oocytes [24, 25]. One of the aims of the present study is to address how MAP kinase phosphorylation is regulated by protein phosphatases. A second purpose is to further clarify the correlation of protein phosphatases or MAP kinase (or both) with chromatin behavior, microtubule organization, and cell cycle progression during pig oocyte maturation.
It has been reported that treatment of 1-cell mouse embryos with OA induces precocious nuclear envelope breakdown of the pronuclei and premature chromosome condensation [26, 27]. The pronuclear disappearance is temporally correlated with MAP kinase phosphorylation induced by OA in fertilized or parthenogenetic mouse eggs [5, 28–31]. Previous work in our laboratory indicates that the sperm microtubule aster fills the cytoplasm during pronuclear movement [32], and that the extensive microtubule organization in fertilized eggs is maintained in the absence of MAP kinase phosphorylation [17]. OA was reported to prevent the events characterizing oocyte activation induced by ionophore A23187 in pig oocytes [33]. How protein phosphatases regulate biochemical and cytological changes in pig oocyte fertilization is largely unknown. The third aim of the present study is to address how protein phosphatases affect biochemical and morphological fertilization events.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro maturation of porcine oocytes was conducted as described by Abeydeera et al. [34]. Briefly, oocytes were aspirated from antral follicles, 3–6 mm in diameter, from ovaries collected from slaughtered prepubertal gilts. After being washed 3 times with Hepes-buffered Tyrode lactate (HTL) containing 0.1% polyvinyl alcohol (PVA), oocytes surrounded by compact cumulus were washed again with tissue culture medium (TCM)-199 (Gibco, Grand Island, NY) supplemented with 0.57 mM cysteine, 10 ng/ml epidermal growth factor (Sigma Chemical Company, St. Louis, MO), and 0.1% PVA. Each group of 50 oocytes was cultured for up to 44 h at 39°C in an atmosphere of 5% CO2 in air in a 500-µl drop of the same medium containing 10 IU/ml FSH (Sigma) and 10 IU/ml hCG (Sigma), with or without drugs.
After maturation culture, oocytes were denuded by pipetting them in maturation medium containing 0.02% hyaluronidase (Sigma). Oocytes cultured in drug-free medium were used for either in vitro fertilization (IVF) or drug treatment. IVF was basically carried out as previously reported [35]. Oocytes were inseminated in a 100-µl drop of modified Tris-buffered medium (mTBM) containing 0.2% BSA and 2 mM caffeine with frozen-thawed ejaculated spermatozoa (5 x 105 cells/ml). Six hours after insemination, oocytes were removed from the fertilization drop and cultured in 500 µl North Carolina State University (NCSU)-23 medium [36] containing 4 mg/ml BSA until examination.
Evaluation of Nuclear Status in Oocytes and Fertilized Eggs by Orcein Staining
Denuded oocytes or fertilized ova were mounted on slides, fixed in acetic acid:alcohol (1:3 v/v) for 24–48 h, stained with 1% orcein, and examined under a phase-contrast microscope.
Immunofluorescent Microscopy of Microtubules
Oocytes or fertilized eggs were fixed with 3.7% paraformaldehyde in PBS for at least 2 h at 4°C. They were first extracted in PBS containing 1% Triton X-100 overnight at 37°C and then blocked in PBS containing 115 mM glycine and 1% Triton X-100 for 30 min. After washing for 15 min, the oocytes were stained with fluorescein isothiocyanate (FITC)-conjugated anti--tubulin antibody (Sigma, for microtubules) diluted 1:50. After 2 washes in PBS, the oocytes were then stained with 10 µg/ml 4',6'-diamidino-2-phenylindole (DAPI). Finally, the oocytes were mounted on glass slides and examined by using a Nikon E600 epifluorescence microscope (Tokyo, Japan).
MAP Kinase Phosphorylation Assay
Proteins from a total of 30 oocytes per treatment were extracted with double-strength electrophoresis buffer. After boiling for 3 min and centrifuging for 3 min at 14 000 x g, the lysates were kept frozen at -80°C until use. Proteins were separated on 10% SDS-polyacrylamide gel for 1 h at 188 V, and then transferred onto Immuobilon-P transfer membrane (Millipore Co., Bedford, MA) for 1 h at 200 mA. The membrane was immersed in methanol for 1 min and dried overnight at room temperature. The membrane was then incubated for 2 h at room temperature with polyclonal rabbit antiphosphorylated MAP kinase antibody (New England BioLabs) diluted 1:600 in PBS containing 5% skim milk pH 7.4. This antibody recognizes both isoforms of phosphorylated MAP kinase, ERK1 and ERK2. After 2 washes for 5 min each in PBS containing 0.01% Tween-20 (PBS-T), the membrane was incubated for 1 h at room temperature with donkey anti-rabbit immunoglobulin G (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1:2000. Finally, the membrane was washed twice in PBS-T for 5 min each time, and then processed by using the enhanced chemiluminescence (ECL) detection system (Amersham International, Amersham, U.K.).
Experimental Design
In experiment 1, GV stage oocytes that had been freshly collected from follicles and were cultured in maturation medium containing 3 µM OA (Sigma). At 5 min, 10, 20, and 30 min, and at 1, 2, 8, 20, 28, or 44 h after culture, samples were collected to examine nuclear status, microtubule assembly (28 and 44 h), and MAP kinase phosphorylation levels, respectively. Oocytes cultured in drug-free medium were used as control.
In experiment 2, GV oocytes were cultured in maturation medium containing 12.5 µM butyrolactone I (BL-I), a purine derivative that specifically inhibits the activation of maturation promoting factor (MPF) by competing with ATP binding to p34cdc2, or 12.5 µM BL-I plus 3 µM OA for 24 h, and GVBD was examined. Oocytes cultured in drug-free medium were used as a control. At 24 h after treatment with BL-I, some oocytes were cultured in media containing 12.5 µM BL-I or 12.5 µM BL-I plus 3 µM OA for another 2 h, and GVBD and MAP kinase phosphorylation were analyzed.
In experiment 3, MII oocytes were exposed to 3 µM OA for 0.5 or 2 h, and spindle morphology was evaluated.
In experiment 4, after 10 h of insemination, pronuclear formation and microtubule organization were evaluated. The presumed fertilized eggs were cultured in the NCSU-23 medium containing 3 µM OA for an additional 0.5 or 2 h, and nuclear status and microtubules were examined. Control eggs were cultured in drug-free medium.
In experiment 5, GV oocytes treated with 3 µM OA for 0.5 or 2 h were washed thoroughly, and then cultured in drug-free medium for up to 44 h to evaluate maturation and microtubule organization. At 10 h postinsemination, the presumed fertilized eggs were treated with 3 µM OA for 0.5 or 2 h, and then, after thorough washing, cultured in NCSU-23 medium for up to 72 h, and cleavage was evaluated. Oocytes or fertilized eggs cultured in drug-free medium were adopted as a control.
Statistical Analysis
The data were analyzed by the chi-square test. Oocytes that had degenerated, including those fragmented, were not included. A value of P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When GV oocytes were cultured in vitro, a low level of MAP kinase phosphorylation was detected at 20 h of culture when 55.6% (30 of 54) of the oocytes went through GVBD. MAP kinase phosphorylation reached the peak level at 28 h of culture when most (77.8%, 28 of 36) of the oocytes reached or went beyond MI stage (Fig. 1). OA greatly accelerated MAP kinase phosphorylation (Fig. 2). When GV oocytes were treated with 3 µM OA, MAP kinase began to be activated as early as 5 min of treatment, and was fully activated by 10 min after treatment. An extremely high level of MAP kinase phosphorylation remained until 8 h of treatment. However, MAP kinase phosphorylation was evidently decreased by 20 h of continuous treatment and little, if any, phosphorylation could be detected at 28 or 44 h (Fig. 2). Meiosis resumption was also greatly accelerated (Table 1).
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Nucleolus disappearance and chromatin condensation were observed in 80.5% (33 of 41) of the oocytes at 2 h of culture, and either chromosomes (86.7%, 52 of 60) or a condensed chromatin cluster (13.3%, 8 of 60) were observed in the GVBD oocytes by 4 h (Fig. 3).
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However, chromosome alignment and spindle organization were inhibited, and meiosis did not go beyond MI stage. Instead, chromosomes were decondensed and were distributed in a large area of the cytoplasm at 28 h (data not shown) or 44 h (Fig. 4) after culture. Microtubule assembly was not observed in most (91.7%, 33 of 36) of the oocytes, although microtubule assembly was detected in a small proportion (8.3%, 3 of 36) of oocytes in the area where the chromosomes were located after 44 h of culture (Fig. 4).
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Okadaic Acid Overcomes the Inhibitory Effect of Butyrolactone I, a Specific MPF Inhibitor, on MAP Kinase Phosphorylation and GVBD
The specific MPF inhibitor, butyrolactone I, at 12.5 µM, significantly blocked GVBD (P < 0.01), and nucleolar changes as well as MAP kinase phosphorylation, when observed at 24 h of culture. GVBD percentages were 78.2% (43 of 55), 14.8% (8 of 54), respectively, in control and BL-I treated groups. Addition of 3 µM OA to the culture medium containing BL-I overcame this inhibitory effect. All the 31 oocytes cultured in the medium containing both BL-I and OA went through GVBD when observed at 24 h of culture, while chromosome alignment was blocked. When oocytes arrested at GV stage by BL-I at 24 h of culture were exposed to OA, all of them (32 of 32) underwent GVBD promptly (within 2 h), and correspondingly, MAP kinase was highly phosphorylated (Fig. 5).
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Okadaic Acid Disrupts Spindle Organization in Metaphase II-Arrested Oocytes
OA induced a dramatic elongation of the MII spindle and disorganization of the metaphase plate when observed 30 min after treatment. An elongated microtubule bundle was still observed 2 h after OA treatment. However, the staining was weaker in most of the oocytes by 2 h of culture (Fig. 6).
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Okadaic Acid Promptly Activates MAP Kinase, Disrupts Microtubules, and Induces Pronuclear Membrane Breakdown in Fertilized Eggs
At 10 h after insemination, pronuclei were formed in 68.5% (63 of 92) of the oocytes and a very low level of MAP kinase phosphorylation was detected (Fig. 7). Extensive microtubule organization was observed around the decondensed sperm head, in the vicinity of the pronuclei, or in the area between the pronuclei in 88.9% (40 of 45) of the pronucleus-stage eggs (Fig. 8). Microtubule organization was also revealed in the second polar body. Treatment of fertilized eggs with OA fully phosphorylated MAP kinase and disrupted microtubules (100%, 31 of 31) by 30 min (Figs. 7 and 8). Pronuclei were still intact, but chromatin was condensed in all 31 fertilized eggs observed by 30 min of OA treatment. By 2 h of OA treatment, pronucleus membrane broke down in most (93.7%, 59 of 63) of the fertilized eggs, and condensed chromatin patches or chromosomes were observed by both orcein staining (data not shown) and fluorescent staining (Fig. 8).
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Effects of OA Treatment Time on Oocyte Maturation and the First Cleavage
As mentioned above, continuous treatment of GV oocytes prevents meiotic cell cycle progression beyond MI stage. When GV oocytes were exposed to OA for 0.5 h, and then, after thorough washing, cultured in drug-free medium, GVBD occurred in 45 out of the 47 oocytes when observed at 4 h of culture, and the second polar body was emitted and normal MII spindle was organized in a large proportion of oocytes when observed at 44 h (Table 2; Fig. 4). In contrast, 2-h treatment prevented oocytes from going beyond MI stage when observed at 44 h (Table 2). In the latter case, MAP kinase was phosphorylated (Fig. 9), microtubules were assembled around the condensed chromosomes, but chromosome alignment was inhibited and an abnormal MI spindle was formed (Fig. 4).
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One-cell embryos cultured continuously in OA-containing medium did not cleave (0%, 0 of 47), whereas half (49.6%, 68 of 137) of the 1-cell embryos incubated with OA for 2 h prior to transfer to OA-free medium cleaved when observed 72 h after insemination.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protein phosphatases 1 and 2A have recently been shown to exist in mouse oocytes, and their relocation and activity changes were detected during oocyte maturation [12, 13]. Activation of MAP kinase by OA has been reported by others and us in mouse and rat oocytes [5–7, 9]. However, all the reports examined MAP kinase changes hours after OA treatment, and thus it is impossible to precisely define the regulation of MAP kinase phosphorylation by protein phosphatases, and their role in the control of meiotic events, because GVBD occurs within a short time after release of oocytes from the follicles in rodents. The present study showed that MAP kinase was promptly phosphorylated (within 10 min) by protein phosphatase inhibition in GV-stage pig oocytes. In contrast, the down-regulation of MAP kinase phosphorylation by PKC activation takes several hours in mouse oocytes [5, 7]. We suggest that inhibition of phosphatases 1 and 2A by OA may directly cause activation of MAP kinase or of the molecules upstream to MAP kinase in the cascade, such as MEK or some other kinases.
The present study showed that GVBD was greatly accelerated following MAP kinase phosphorylation induced by OA. MAP kinase phosphorylation first stimulated chromatin condensation and nucleolus disappearance, and then disruption of GV membrane. Two kinases, MPF and MAP kinase, play a pivotal role in oocyte maturation. Depending on the species, MAP kinase is activated either after MPF activation [3–7] or at approximately the same time of MPF activation during normal oocyte maturation [37]. Recent studies show that MAP kinase activation is uncoupled from MPF activation in Xenopus or bovine oocytes [38, 39]. Previous studies show that OA accelerates both MPF activation and meiotic resumption in Xenopus [40] and bovine oocytes [18], whereas other studies show that OA accelerates MAP kinase activation, but partially inhibits MPF activation in rat oocytes [6]. In addition, only MAP kinase was activated when OA was used to induce GVBD of mouse oocytes arrested at the GV stage by 3-isobutyl-1-methylxanthine (IBMX) [41]. Furthermore, MAP kinase rather than p34cdc2 kinase induces chromosome condensation and precocious GVBD in maturation-incompetent mouse oocytes [10, 42]. When comparing our results with previous studies [10], we found that MPF activation was detected later than MAP kinase activation in pig oocytes exposed to OA. We also show that activation of MAP kinase by protein phosphatase inhibition greatly accelerated GVBD when MPF activity was inhibited by the specific inhibitor BL-I. BL-I specifically inhibits cdc2 kinase by competing with ATP, not with the substrate, and its IC50 value against cyclin B-cdc2 kinase (0.68 mM) is 140 times lower than that against MAP kinase [43], and previous studies show that OA is able to induce pig oocyte GVBD without activation of histone H1 kinase [44]. We thus conclude that induction of pig oocyte GVBD by OA may be due to MAP kinase activation rather than MPF activation.
The stimulation of meiosis resumption does not require the continuous presence of OA. Half-hour treatment of GV oocytes induces GVBD at the same time course as continuous treatment. In fact, MAP kinase is activated within 10 min, and 30-min treatment already causes some cytoplasmic abnormalities that lead to some oocytes failing to go beyond MI stage, although a large percentage of the oocytes are able to complete maturation. It has been reported that treatment of rhesus macaque oocytes with 1 µM OA enhances GVBD rate without causing cytoplasmic abnormalities [45]. The disruption of GV oocyte cytoplasm by long-term (2 h) OA treatment is not reversible. In these oocytes, the MI spindle is not properly organized, although MAP kinase is phosphorylated, and microtubules are organized in the chromosome area when observed at 44 h of culture. This may be caused by disruption of the capture of microtubules by chromosome kinetochores, or by the organization of microtubules by centrosomes, or both, as in pig oocytes treated with taxol [25]. We also showed that MAP kinase phosphorylation caused by inhibition of protein phosphatases reached a low level at 20 h, and correspondingly, microtubule organization was inhibited. Instead, chromatin decondensed and dispersed in the cytoplasm. These results provide further evidence for the critical role of MAP kinase phosphorylation in spindle microtubule assembly and spindle formation. In the presence of OA, MPF activity also drops progressively 2 h after GVBD in rat oocytes [6]. OA induced spindle elongation and metaphase plate disorganization in MII-arrested pig oocytes. The same phenomenon is also seen in MII-arrested mouse oocytes [15, 16]. This may be caused by hyperphosphorylation of some proteins. In rodent oocytes, OA induced hyperphosphorylation of several proteins associated with the spindle, including MAP kinase [7, 16].
We recently reported that MAP kinase is still in a phosphorylated form 3.5 h after insemination, and it is dephosphorylated when pronuclei formed [17]. Here we further prove that inhibition of protein phosphatases promptly causes MAP kinase phosphorylation, and, after a delay, chromosome condensation and pronucleus membrane disruption. Like inhibition of polar body emission during oocyte maturation, OA treatment also caused a time-dependent inhibition of cleavage of fertilized eggs. This is consistent with the results obtained in mouse eggs [26]. It appears that MAP kinase phosphorylation is incompatible with the presence of a pronuclear envelope in pig eggs, as in mouse eggs.
In farm animals, including pigs, microtubules assemble around the base of the incorporated sperm heads, and as development continues, microtubules expand and surround the pronuclei, participating in pronuclear apposition [32, 46]. We recently reported that the assembly of microtubules around the pronuclei occurs in the absence of MAP kinase phosphorylation [17]. Here we further prove that stimulation of MAP kinase phosphorylation by OA promptly disassembles interphase microtubules.
Microtubules are nucleated by maternal centrosomes in fertilized mouse eggs, whereas microtubules are dominantly assembled by paternally contributed centrosomes in domestic species. Furthermore, fertilization mechanisms in cows are more similar to lower animals than to the mouse (for review see [46]). Recent studies have shown that MAP kinase may suppress sperm centrosome function in nucleating microtubule asters, while keeping the spindle integrity in the same cytoplasm in a variety of lower animal (Urechis caupo, oyster, and starfish) eggs fertilized in prophase [47, 48]. On the basis of previous reports and our present findings, we suggest that MAP kinase may suppress microtubule assembly by sperm centrosomes in fertilized pig eggs.
In summary, the present study shows that 1) inhibition of protein phosphatases by OA promptly stimulates MAP kinase phosphorylation in GV oocytes and fertilized eggs; 2) OA may stimulate meiosis resumption by MAP kinase activation; 3) long-term exposure of oocytes to OA causes MAP kinase dephosphorylation and spindle disorganization, and thus blocks meiosis progression; and 4) MAP kinase may be a suppressor of microtubule assembly by sperm centrosomes in fertilized pig eggs.
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FOOTNOTES |
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1 This study was supported by grants from the University of Missouri-Columbia to H.S. and Food for the 21st Century to R.S.P. Q.Y.S., a research associate working in the laboratory of H.S., is supported by the Special Funds for Major State Basic Research ("973") project (G1999055902) while working at the University of Missouri. This manuscript is a contribution from the Missouri Agriculture Experiment Station, journal series 13151. 
2 Correspondence: Heide Schatten, Department of Veterinary Pathobiology, W137 Veterinary Medicine Building, University of Missouri-Columbia, 1600 East Rollins, Columbia, MO 65211. FAX: 573 884 5414; schattenh{at}missouri.edu
Accepted: October 9, 2001.
Received: July 9, 2001.
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-tubulin, and MAP kinase. Mol Reprod Dev 2001; 60:481-490[CrossRef][Medline]This article has been cited by other articles:
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  C.-G. Liang, Y.-Q. Su, H.-Y. Fan, H. Schatten, and Q.-Y. Sun Mechanisms Regulating Oocyte Meiotic Resumption: Roles of Mitogen-Activated Protein Kinase Mol. Endocrinol., September 1, 2007; 21(9): 2037 - 2055. [Abstract] [Full Text] [PDF]
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