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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, China
ABSTRACT
The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.
fertilization, in vitro fertilization, meiosis, oocyte development, ovum
INTRODUCTION
During mammalian oocyte maturation and fertilization, many dynamic events occur to ensure the successful meiotic divisions and the accurate union of the parental genomes. The cascade of events includes the resumption of meiosis, chromosome condensation, spindle formation and migration, polar body (PB) emission, cortical granule migration, anchoring at the cortex and exocytosis, sperm incorporation, pronuclear (PN) formation, and syngamy. The organization of the cytoskeleton, in particular microtubules and microfilaments, is well known to be involved in the regulation of these dynamic events in many lower animals such as sea urchins and Xenopus [1, 2]. The vast majority of information in mammals has been derived from studies on rodents, particularly the mouse. It has been revealed that both microtubules and microfilaments play a critical role in the chromosomal and cytoplasmic dynamic events during oocyte maturation and fertilization. However, accumulating information from the studies on this subject for other species suggests that rodents may be atypical with regard to cytoskeletal functions during oocyte maturation and fertilization [3]. For example, microtubule organization and configuration in the cytoplasm of bovine oocytes before and after fertilization are different from mouse oocytes, and cows may undergo fertilization mechanisms more similar to lower vertebrates such as the frog than the mouse [4, 5].
In the pig, Kim et al. [6–8] and Wang et al. [9] have studied microtubule and microfilament organization and dynamics during oocyte maturation, aging, parthenogenetic activation, and fertilization. Kim et al. [10] also showed that microfilaments are essential for cortical granule (CG) migration to the cortex during oocyte maturation. Furthermore, Kim et al. [8] demonstrated that inhibition of either microfilament or microtubule assembly affects cytokinesis but not PN formation during in vitro fertilization. However, the involvement of microtubule and microfilament cytoskeleton in many other important events of oocyte maturation and fertilization has not been addressed in this species.
In mammalian species studied so far the kinetics of maturation promoting factor (MPF) and mitogen-activated protein (MAP) kinase, the two important protein kinases that regulate meiotic cell cycle, are different during oocyte maturation and fertilization [11]. A recent study showed that MAP kinase plays a more important role in controlling chromatin and microtubule behavior than MPF in mouse oocytes [12]. Activation of MAP kinase keeps microtubules and chromatin from entering interphase configuration [11]. However, MAP kinase phosphorylation in relation to cytoskeletal behavior during porcine oocyte maturation and fertilization has not been studied.
We undertook the present study to clarify the role of microfilaments and microtubules in several important dynamic events of porcine oocyte maturation and fertilization including CGs and spindle anchoring at the cortex, CG exocytosis, sperm penetration, and PN syngamy. We also investigated MAP kinase phosphorylation in relation to nuclear and cytoskeletal behavior during oocyte maturation and fertilization.
MATERIALS AND METHODS
In Vitro Maturation and Fertilization of Oocytes
In vitro maturation of porcine oocytes was conducted by the method described by Abeydeera et al. [13]. Briefly, oocytes were aspirated from antral follicles, 3–6 mm in diameter, of ovaries collected from slaughtered prepubertal gilts. After being washed three times with Hepes-buffered Tyrode lactate containing 0.1% polyvinyl alcohol (HTL-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 Co., 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 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. The IVF was carried out by the method reported previously with very minor modifications [14]. 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 (3 x 105 cells/ml) that were previously preincubated for 3 h in the same medium. Three hours after insemination, oocytes were removed from the fertilization drop and cultured in 500 µl North Carolina State University (NCSU)-23 medium [15] containing 4 mg/ml BSA until examination.
Evaluation of Sperm Penetration and Nuclear Status
Denuded oocytes or fertilized ova were mounted on slides, fixed in acetic acid alcohol (1:3, v:v) for 24 h, stained with 1% orcein, and examined under a phase-contrast microscope. For immunofluorescence examination, DNA was stained with 10 µg/ml of either propidium iodine (PI) or 4',6-diamidino-2-phenylindole (DAPI) for 5–10 min and observed with a Nickon epifluorescence microscope or a BioRad 600 laser scanning confocal microscope (BioRad, Hercules, CA).
Confocal Microscopy of CGs
Cortical granule detection was based on the procedures reported in our previous study [16]. For assessment of CG migration, cumulus-free, zona-intact oocytes were used. For assessment of CG anchoring and exocytosis, the zonae pellucidae were removed by treating the oocytes with 0.05% pronase (Sigma) in PBS. Oocytes were fixed with 3.7% paraformaldehyde in PBS for 1 h at room temperature, followed by blocking in PBS containing 0.3% BSA (Sigma) and 100 mM glycine three times, for 5 min each. After permeabilization for 5 min in PBS containing 0.1% Triton X-100 (Sigma), oocytes were washed two additional times for 5 min each in blocking solution. They were then cultured in 200 µM fluorescein isothiocyanate (FITC)-labeled peanut agglutinin (PNA) (Sigma) in PBS for 30 min in the dark. Finally, the oocytes were washed three times in PBS containing 0.3% BSA and 0.01% Triton X-100. The DNA was stained in the final rinse with PBS containing 10 µg/ml PI. Oocytes were mounted on glass slides and observed with a laser scanning confocal microscope.
Examination of Microtubules and Microfilaments
Oocytes 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 for 2 h at 37°C or overnight at room temperature and then blocked in PBS containing 115 mM glycine and 1% Triton for 30 min. After washing for 15 min, the oocytes were stained with 1 µg/ml FITC-phalloidin (Sigma; for actin microfilaments) or FITC-conjugated anti-
-tubulin antibody (Sigma; for microtubules) diluted 1:50. After two washes in PBS, the oocytes were then stained with PI or DAPI. Finally, the oocytes were mounted on glass slides and examined by using either a Nikon E600 epifluorescence or a BioRad 600 laser scanning confocal microscope.
Mitogen-Activated Protein 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 a 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 anti-phosphorylated MAP kinase antibody (New England Biolabs, Beverly, MA) diluted 1:600 in PBS containing 5% skimmed milk (pH 7.4). This antibody recognizes both isoforms of phosphorylated MAP kinase, ERK1 and ERK2. After two washes, 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 IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1:2000. Finally, the membrane was washed twice in PBS-T, 5 min each, and then processed by using the ECL detection system (Amersham International, Little Chalfont, Buckinghamshire, UK).
Experimental Design
Experiment 1 Germinal vesicle (GV) stage oocytes were cultured in the maturation media containing different concentrations of microfilament polymerization inhibitor, cytochalasin B (CB; Sigma), or microtubule polymerization inhibitor, nocodazole (Sigma), for 44 h. Chromatin state and position as well as spindle formation and migration were evaluated. Oocytes cultured in drug-free medium for 44 h were treated with 200 µM CB for 1.5 h to observe the orientation and anchorage of their spindles.
Experiment 2 The GV oocytes were cultured in maturation media containing different concentrations of CB or nocodazole for 44 h, and then the CG distribution was evaluated. The role of microtubule and microfilament cytoskeleton in maintaining the anchorage of CGs at the cortex was examined by treating the zona-free metaphase II (MII)-arrested oocytes for 1.5 h or 3 h with either CB or nocodazole and then determining the localization of the CGs. To determine whether the microtubule and microfilament cytoskeleton is involved in CG exocytosis during oocyte activation, zona-free MII-arrested oocytes were first treated with CB and then incubated with spermatozoa. Because it was found that microfilament disruption inhibited sperm incorporation (shown below) and microtubule disruption is expected to influence sperm and thus inhibit penetration, an electrical pulse of 120 V, 31 µsec was applied to CB- or nocodazole-treated oocytes, and CG exocytosis was examined 1.5 h after stimulation. To exclude the possibility that electrical pulse-induced CG exocytosis in CB-treated oocytes is caused by electroporation of membrane, the CB-treated zona-free oocytes were activated by treatment with 50 µM ionophore A23187 for 5 min, 100 nM phorbol 12-myristate 13-acetate (Sigma) for 1 h, or 200 µM thimerosal for 30 min and CG exocytosis was evaluated 1 h later.
Experiment 3 To detect the function of microfilaments in sperm penetration during fertilization, oocytes matured in vitro for 44 h were incubated with CB for 1.5 h and then inseminated with spermatozoa for 5 h for evaluation of sperm penetration. Another approach was to inseminate CB-treated oocytes with spermatozoa that had been preincubated for 3 h. Two and a half hours after insemination, the oocytes were transferred into CB-free NCSU-23 medium and cultured for 18 h to evaluate PN formation. To exclude the possibility that the effect of microfilaments on fertilization is caused by inhibiting PN formation, CB-treated oocytes were activated by a single electric pulse of 120 V, 31 µsec, and PN formation was evaluated 6 h after activation. To determine the role of the microtubules and microfilaments in syngamy, mature oocytes were inseminated with preincubated spermatozoa for 3 h to guarantee sperm penetration, and they were then cultured in NCSU-23 media containing either 200 µM CB or 10 µM nocodazole for 20 h. Pronuclear status and apposition as well as second PB (PB2) emission were evaluated. Cleavage was recorded 44 h after insemination.
Experiment 4 Oocytes cultured in vitro for 0, 8, 20, 24, 30, 44, and 76 h for maturation and those collected 0, 3.5, 10, and 20 h postinsemination were used for microtubule and chromatin observation and pooled for detection of MAP kinase phosphorylation by Western blot. The GV oocytes matured in the media containing different concentrations of CB or nocodazole for 44 h, and mature oocytes treated with 1 µM taxol, a drug that induces extensive cytoplasmic aster formation, for 10 min were used for evaluating the relationship between cytoskeletal organization and MAP kinase phosphorylation.
Statistical Analysis
The data were analyzed by 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.
RESULTS
Role of Microfilaments and Microtubules in Chromosome and Spindle Formation, Movement, and Anchorage to the Cortex and Chromosome Separation
During oocyte maturation, microfilaments were concentrated to the domain where the MI spindle was located and the chromosomes separated (Fig. 1, A–C). They were uniformly distributed in the cortex, with a thickening over the meiotic spindle in MII oocytes (Fig. 1D). Nocodazole treatment did not influence microfilament distribution, while culture of GV oocytes with 200 µM CB for 44 h or treatment of MII oocytes with 200 µM CB for 1.5 h effectively blocked microfilament assembly (Fig. 1, E and F). As shown in Table 1, microfilament disassembly by treatment of oocytes with high concentrations (200 µM, 2000 µM) of CB did not influence GV breakdown, chromosome condensation, or MI spindle formation but significantly inhibited further progression of the first meiotic division when compared to oocytes cultured in the drug-free medium or in the medium containing low concentrations (2 µM, 20 µM) of CB (P < 0.01). The MII spindles were located in the cortex and oriented radially to the cell surface in most of the mature oocytes as demonstrated by anti--tubulin indirect immunofluorescence microscopy (Fig. 2, A and B). In a few oocytes, MII spindles were anchored to the cell cortex tangentially (Fig. 2C). When the CB concentration was increased to 200 µM the MI meiotic apparatus formed, but its localization at the cell cortex was blocked (Table 1; Fig. 2, D–F). Oocytes incubated in nocodazole did not form meiotic spindles, however, GV breakdown occurred and chromosomes condensed and translocated to the cortex (Table 2; Fig. 2, G–I). Once the MII spindle was anchored radially to the cell cortex disruption of microfilaments by treating the oocytes with 200 µM CB for 1.5 h influenced neither its orientation nor its cortical localization (Fig. 2J). Of the 22 CB-treated oocytes, 19 had spindles radially localized at the cortex. Treatment of MII oocytes with 1 µM taxol for 10 min distorted spindle morphology in all oocytes. Microtubules were assembled in the vicinity of the spindle, and cytoplasmic asters were induced (Fig. 2, K and L).
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Effects of Microfilament and Microtubule Inhibitors on CG Migration and Anchorage at the Cortex During Oocyte Maturation and CG Exocytosis During Oocyte Activation
The CGs were distributed in the entire cytoplasm or entire cortex of an oocyte at the GV stage. During oocyte maturation, the CGs migrated peripherally, assuming a position just beneath the plasma membrane. The dependence of the peripheral CG migration on microtubule and microfilament cytoskeleton assembly was investigated. The arrangement of CGs was designated as migrated as shown in Figure 3A and nonmigrated as shown in Figure 3, B and C. We found that microfilaments rather than microtubules controlled the peripheral CG migration during oocyte maturation. When high concentrations (200 µM, 2000 µM) of CB were present during oocyte maturation, CG migration was blocked in all the oocytes examined, while CG migration occurred in all the oocytes treated with nocodazole.
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We next determined whether microfilaments and microtubules were required for the anchoring of CGs to the oolemma. The MII oocytes were treated with either 200 µM CB or 10 µM nocodazole for 1.5 or 3 h to disrupt microfilaments and microtubules, and CG distribution was determined. Microtubule disruption did not influence CG anchoring. When oocytes were treated with CB, microfilaments were disrupted, but there was no apparent CG inward migration or precocious CG exocytosis (Fig. 3D).
We treated MII oocytes with CB for 1.5 h and then incubated oocytes with spermatozoa that had been preincubated for 3 h in order to determine whether microfilament disruption affects CG exocytosis during oocyte activation induced by sperm. However, sperm penetration was inhibited by microfilament disruption as stated below. Thus, we activated CB-treated oocytes with an electrical pulse and found that inhibition of microfilament assembly did not block CG exocytosis (Fig. 3E). In a few oocytes, inward CG movement was observed (Fig. 3, F and G). When nocodazole was used, similar results were obtained. Because an electrical pulse can induce cell plasma membrane poration we also confirmed this finding by activating CB-treated oocytes with ionophore A23187, thimerosal, or phorbol ester to exclude the possibility that CG exocytosis in CB-treated oocytes was due to the electrical portion. We examined more than 30 oocytes for each treatment and found that all the CB-treated oocytes showed complete or partial CG exocytosis after activation with each of the three methods. A23187 and phorbol ester stimulated CG exocytosis but not inward movement (Fig. 3, F, H, and I), while thimerosal, like electrical stimulation, induced both CG exocytosis and inward movement (Fig. 3, K and L).
Effects of Microtubule and Microfilament Cytoskeleton Disassembly on Sperm Incorporation and PN Formation, Apposition, and Syngamy
To evaluate the effects of microfilament disruption on sperm penetration, oocytes were treated with different concentrations of CB for 1.5 h and then inseminated with spermatozoa. At 5 h postinsemination 83.1% (69 of 83) of the oocytes were penetrated, of which 79.7% (55 of 69) contained more than two sperm heads in the control group. In contrast, 87.9% (58 of 66) of the CB-treated oocytes were not penetrated and polyspermy was found only in two ova. Inhibition of sperm penetration was also proven by the fact that PN formation was significantly decreased in CB-treated oocytes after insemination (P < 0.01, Table 3), while PN formed in most of the oocytes that were activated by an electrical pulse, although PB2 emission was inhibited (Table 4).
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When MII oocytes were incubated with spermatozoa preincubated for 3 h to assure sperm penetration and then cultured in medium containing either CB or nocodazole for 20 h, PN were observed in 40% of the ova, suggesting that neither microfilament nor microtubule assembly inhibition affects PN formation (Table 5). However, male and female PN centration took place in CB-treated ova (Fig. 4A), but not in nocodazole-treated ones. In unfertilized oocytes treated with nocodazole, sperm were found on the surface of zona pellucida, and clusters of chromosomes were distributed in the cytoplasm. At 23 h postinsemination, breakdown of the PN envelopes and condensation of the maternally and paternally derived chromosomes occurred in some nocodazole-treated fertilized ova, even though the close apposition of PN did not take place (Fig. 4B). In contrast, mitotic chromosomes began to appear in some of the control or CB-treated fertilized ova. Cytokinesis was inhibited in both CB- and nocodazole-treated fertilized ova when observed 48 h postinsemination (Table 5).
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Mitogen-Activated Protein Kinase Phosphorylation in Relation to Chromatin and Cytoskeleton Organization
Oocytes matured in vitro for 8 h were still at the GV stage, and MAP kinase was not phosphorylated. At 20–24 h of culture 60% (36 of 60) of the oocytes proceeded to or beyond the MI stage, and MAP kinase was phosphorylated. At 30 h of culture when most of the oocytes were at stages from MI to MII, MAP kinase phosphorylation reached the peak level as in MII oocytes 44 h after culture (Fig. 5). At 3.5 h after insemination when most of the oocytes still have condensed chromosomes and when microtubules were not in an interphase configuration, MAP kinase was in a phosphorylated form. However, MAP kinase was dephosphorylated 10 h after insemination when PN formed (Fig. 6). The MAP kinase was completely dephosphorylated 20 h after insemination (Fig. 6), while in unfertilized oocytes MAP kinase was still in a phosphorylated form 28 h after maturation (72 h after culture) (Fig. 5). Chromosome condensation and metaphase microtubule assembly were temporally correlated with MAP kinase phosphorylation, while the transition of condensed chromosomes into interphase and metaphase microtubule disappearance were consistent with MAP kinase dephosphorylation. In oocytes first treated with CB and then inseminated, MAP kinase was still in a phosphorylated form, providing further evidence for the inhibition of sperm penetration by microfilament disruption (Fig. 7). When oocytes were cultured in medium containing CB, both chromosome condensation and spindle formation occurred as described above and MAP kinase was phosphorylated. When oocytes were cultured with nocodazole, chromosome condensation and MAP kinase phosphorylation occurred, although spindle formation was inhibited (Fig. 8). Treatment of either MI or MII oocytes with 1 µM taxol for 10 min, although inducing changes in microtubule organization, did not increase MAP kinase phosphorylation levels (Fig. 9).
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The effects of microtubule and microfilament inhibitors on the events of porcine oocyte maturation and fertilization are summarized in Table 6.
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DISCUSSION
Although recent reports reveal that polymerization of both microtubules and microfilaments is important for porcine oocyte maturation and fertilization [6–8, 10], the role of the cytoskeleton in many important dynamic events is not known. We presented here several new findings on the role of microtubules and microfilaments during porcine oocyte maturation and fertilization. In addition, the correlation between MAP kinase phosphorylation and chromatin and cytoskeletal changes was studied.
Changes in cytoskeletal configuration have been implicated in the progression of meiosis. In the rat, GV breakdown is reversibly inhibited by a microtubule-disrupting agent, nocodazole [17]. However, neither CB nor colchicine inhibited GV breakdown and chromosome condensation during mouse oocyte maturation [18]. A similar phenomenon was observed in porcine oocytes in which GV breakdown and chromosome formation were not blocked by either microfilament or microtubule disruption. However, further meiotic progression did not occur. Morphologically normal MI spindles were formed in CB-treated oocytes, but the formation of a microfilament domain around the spindle and localization of the spindle at the cortex were inhibited in most of the oocytes. It has also been reported that spindle migration to the cell cortex is driven by microfilaments, and CB-treated mouse oocytes maintain an intact meiotic MI spindle [18]. In the domestic species, peripheral positioning of the germinal vesicle next to the oocyte cortex may occur prior to GV breakdown [3]. Thus, the positioning of MI spindle in the interior cytoplasm of CB-treated oocytes may be the result of inhibition of GV movement by microfilament disruption. These observations indicate that microfilaments are not involved in the early phase of meiosis I (including GV breakdown, chromosome condensation, MI spindle formation), while the cortical positioning of the MI spindle and further progression of meiosis I are microfilament dependent in porcine oocytes. This study also revealed that proper orientation and anchorage of the MII spindle are independent of microfilaments. A recent study shows that proper positioning of the mitotic spindle along a particular axis at the cell cortex is mediated by a cortical microtubule capture mechanism in which the microtubule-binding factor family, Bim1, directly interacts with cell surface-associated proteins such as Kar9 [19].
Microtubules play roles distinct from microfilaments during oocyte maturation. After incubation of oocytes with nocodazole the spindle did not form and the progression of meiosis I was blocked. However, the chromosomes were condensed, the chromosome cluster was positioned at the cell cortex, and actin filament clustering occurred in the vicinity of chromosomes.
During mammalian oocyte growth and maturation, CGs migrated toward the cortex, assuming a position adjacent to the plasma membrane. Previous work indicates that, in pig oocytes as in mouse oocytes, CG movement to the cortex depends on microfilament assembly [10, 20]. Our experiments also showed that microfilaments rather than microtubules control CG migration to the cortex beneath the oolemma. Once CGs are established in the cortex following oocyte maturation, they are anchored there and unable to move back into the interior [20]. In order to determine whether microfilaments or microtubules play any role in maintaining CGs at the cortex, mature oocytes were treated with either CB or nocodazole for 1.5 or 3 h. Microfilaments or microtubules were disrupted, but CGs were still anchored to the oolemma, suggesting that neither microfilaments nor microtubules are required for maintaining the CG anchorage at the cortex. Similar findings were also recently obtained in mouse oocytes [20].
Sperm-egg fusion induces an increase in intracellular free calcium and exocytosis of CGs, which results in a block to polyspermy. In mouse and hamster it has been well established that intact microfilaments are required for normal CG exocytosis induced by sperm [21–23]. However, unlike rodent oocytes, sperm penetration was inhibited when microfilaments were disrupted as revealed in this study. Thus, we used various artificial stimulation [16, 24–26] to find out if microfilaments are necessary for CG exocytosis. All the chemical and physical stimuli were found to effectively induce CG exocytosis in oocytes pretreated with CB for 1.5 or 3 h. Migration of some CGs to the interior ooplasm was also observed in electrical-stimulated or thimerosal-treated oocytes. These results suggest that CG exocytosis is independent of microfilament assembly in the cell cortex and that different stimuli may induce CG exocytosis by different mechanisms. In zebrafish oocytes, subplasmalemmal filamentous (F) actin acts as a physical barrier to CG secretion and is locally disassembled prior to granule release. Cortical granule exocytosis is accelerated in CB- or cytochalasin D-treated oocytes [27, 28]. When porcine oocytes were treated with a microtubule inhibitor, nocodazole, the cortical reaction also occurred. Therefore, we conclude that CG exocytosis depends on neither microfilaments nor microtubules in porcine oocytes.
Microfilaments and microtubules control a variety of events during fertilization. The present study indicates that the pig may undergo fertilization mechanisms more similar to lower vertebrates than to the mouse. In many lower animals such as sea urchins [1], Pelvetia [29], and starfish [30, 31], sperm incorporation into the oocyte is inhibited by microfilament inhibitors. Microfilament inhibitors were also reported to inhibit sperm incorporation in bovine oocytes [32]. In contrast, microfilaments do not interfere with sperm entry into mouse oocytes [22, 33, 34]. Our results show that sperm penetration is dependent on microfilament assembly in porcine oocytes. This was evidenced by the fact that sperm penetration and PN formation was significantly decreased when the oocytes were treated with CB before coincubating with sperm, while PN formed in most of the CB-treated oocytes after electrical stimulation. The dephosphorylation of MAP kinase did not occur by 20 h after insemination in CB-treated oocytes, while the kinase was completely dephosphorylated at this time in the fertilized control group, providing further evidence for the blockage of sperm penetration in CB-treated oocytes.
Unlike mouse oocytes, in which microtubule assembly is required for PN formation [35, 36], neither microtubule nor microfilament disruption affected PN formation in porcine oocytes. This is consistent with our previous findings obtained in sea urchin eggs [37]. The requirement for microtubules and microfilaments for PN migration is different in different animals. In mouse oocytes, PN formation occurs normally when microfilament assembly is inhibited, but PN centration does not proceed [3, 23, 36]. In contrast, microtubule assembly is required for PN movement in fertilized ova of a variety of other animals including sea urchins [37], Pelvetia [29], Beroe ovata [38], Caenorhabditis elegans [39], rabbit [40], sheep [41], and cows [5]. We demonstrated that PN moved and the first mitotic spindle formed in CB-treated fertilized pig zygotes, while close apposition of the PN did not take place in nocadazole-treated fertilized ova. Pronuclear envelope breakdown took place and two separate clusters of chromosomes derived from male and female PN formed. We propose that microtubule rather than microfilament functions facilitate the syngamy of the PN in porcine fertilization.
It has been previously reported by us and others that MAP kinase phosphorylation in mouse oocytes occurs only after GV breakdown and remains at a high level from MI to MII stage when chromosomes are condensed, and microtubules are not in an interphase configuration [11, 42]. An upstream activator of MAP kinase, MOS, is also proven to be responsible for the control of microtubule and chromatin organization. Mitogen-activated protein kinase failed to be activated in c-mos knockout mouse oocytes, and abnormal diffused spindles, loosely condensed chromosomes, and large polar bodies were observed, although p34cdc2 kinase activity is normal [43–46]. It is further proposed that MAP kinase but not MPF controls chromatin and microtubule behavior in mouse oocytes [12]. By using an anti-phosphorylated MAP kinase antibody, we showed in this paper that MAP kinase was phosphorylated in oocytes matured for 20–24 h when 60% of the oocytes proceeded to MI. It reached the peak level at 30 h when most of the oocytes were at MI–MII and remained high until 44 h. Inoue et al. [47] used a different culture system, in which oocyte meiotic maturation progressed slower than in the system utilized in this study, to evaluate MAP kinase phosphorylation by in-gel assay and found that MAP kinase was not phosphorylated up to 25 h of culture, but phosphorylation significantly increased at 30 h when 56% of the oocytes proceeded to or beyond MI. The present study reveals for the first time that MAP kinase is still present in a phosphorylated form 3.5 h after insemination when chromatin and microtubules had not entered interphase, while it was dephosphorylated when PN formed. All the evidence suggests that, as in mouse oocytes, MAP kinase phosphorylation in porcine oocytes may be involved in keeping the chromatin and microtubule organization from entering interphase and assure the progression of meiosis. However, our studies revealed that MAP kinase phosphorylation does not directly control the progression of meiosis from MI to MII. Cytochalasin B-treated oocytes were arrested at the MI stage, although MAP kinase was fully phosphorylated, and normal spindles were formed. Our study also showed that microtubule assembly did not occur in the presence of nocodazole even though the MAP kinase was phosphorylated. On the other hand, microtubule aster formation induced by taxol occurred while the level of MAP kinase phosphorylation did not change in MII oocytes. Another interesting finding was that MAP kinase remained dephosphorylated 10–20 h postinsemination when microtubules are fully assembled into the sperm aster complex for PN apposition [8]. Thus, microtubule organization by the sperm aster, which is required for PN apposition and syngamy, is not dependent on MAP kinase phosphorylation.
In conclusion, meiosis progression of porcine oocytes does not go beyond the MI stage when microtubules or microfilaments are disrupted, even though MAP kinase is phosphorylated. Migration of CGs to the cortex is controlled by microfilaments rather than microtubules, but peripheral anchorage and exocytosis of CGs are not affected by disruption of either cytoskeleton. Microfilaments are involved in sperm penetration, but microtubules play a role in syngamy during fertilization. Phosphorylation of MAP kinase, which may be involved in spindle assembly and chromatin condensation, is not required for microtubule organization by the sperm aster.
FOOTNOTES
First decision: 6 September 2000.
1 This study was supported by grants from the University of Missouri-Columbia to H.S., Food for the 21st Century Program to R.S.P. Q.Y.S., a research associate working in H.S.'s lab, is supported by the "973" project (G1999055902) while working at the University of Missouri. This manuscript is a contribution from the Missouri Agriculture Experiment Station, journal series no. 13051. 
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 31, 2000.
Received: July 21, 2000.
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T. A.L. Brevini, R. Vassena, C. Francisci, and F. Gandolfi Role of Adenosine Triphosphate, Active Mitochondria, and Microtubules in the Acquisition of Developmental Competence of Parthenogenetically Activated Pig Oocytes Biol Reprod, May 1, 2005; 72(5): 1218 - 1223. [Abstract] [Full Text] [PDF]
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E Eliyahu, A Tsaadon, N Shtraizent, and R Shalgi The involvement of protein kinase C and actin filaments in cortical granule exocytosis in the rat Reproduction, February 1, 2005; 129(2): 161 - 170. [Abstract] [Full Text] [PDF]
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K. Miyoshi, S. J. Rzucidlo, S. L. Pratt, and S. L. Stice Improvements in Cloning Efficiencies May Be Possible by Increasing Uniformity in Recipient Oocytes and Donor Cells Biol Reprod, April 1, 2003; 68(4): 1079 - 1086. [Abstract] [Full Text] [PDF]
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Z.-Y. Zhu, D.-Y. Chen, J.-S. Li, L. Lian, L. Lei, Z.-M. Han, and Q.-Y. Sun Rotation of Meiotic Spindle Is Controlled by Microfilaments in Mouse Oocytes Biol Reprod, March 1, 2003; 68(3): 943 - 946. [Abstract] [Full Text] [PDF]
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H.-Y. Fan, C. Tong, L. Lian, S.-W. Li, W.-X. Gao, Y. Cheng, D.-Y. Chen, H. Schatten, and Q.-Y. Sun Characterization of Ribosomal S6 Protein Kinase p90rsk During Meiotic Maturation and Fertilization in Pig Oocytes: Mitogen-Activated Protein Kinase-Associated Activation and Localization Biol Reprod, March 1, 2003; 68(3): 968 - 977. [Abstract] [Full Text] [PDF]
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B. A. McAvey, G. B. Wortzman, C. J. Williams, and J. P. Evans Involvement of Calcium Signaling and the Actin Cytoskeleton in the Membrane Block to Polyspermy in Mouse Eggs Biol Reprod, October 1, 2002; 67(4): 1342 - 1352. [Abstract] [Full Text] [PDF]
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