Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 61, 111-119 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Effect of Myosin Light Chain Kinase, Protein Kinase A, and Protein Kinase C Inhibition on Porcine Oocyte Activation1

Kristin M. Greena, Jae Hwan Kima, Wei-Hua Wanga, Billy N. Daya, and Randall S. Prather2,a

a Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have shown that exposure to broad-spectrum protein kinase inhibitors results in parthenogenetic activation of metaphase II arrested porcine oocytes. The objective of this study was to determine the effect of inhibitors of myosin light chain kinase and other protein kinases on pronuclear development, dephosphorylation of a 25-kDa protein, and cortical granule exocytosis. Metaphase II arrested oocytes were obtained by in vitro maturation. Cumulus-free oocytes were cultured with specific inhibitors in modified Whitten's medium for 24 h. Treatment with inhibitors that should inhibit myosin light chain kinase—HA100 (250 µM), Wortmannin (1 µM), and a combination of Wortmannin (1 µM), KT5720 (75 nM), and Iso-H7 (50 µM)—resulted in significantly higher pronuclear development (74.0%, 18.0%, and 35.0%, respectively) than in the negative control, H7 (10 µM; 2.0–12.4% depending upon the replication). Treatment with HA100 (250 µM) resulted in the dephosphorylation of the 25-kDa protein to a 22-kDa protein in 80.0% (n = 10) of oocytes exposed. However, Wortmannin (1 µM; n = 17), KT5720 (75 nM; n = 16), and Iso-H7 (50 µM; n = 19) treatment individually and in combination (n = 19) did not result in significant (p < 0.05; n = 19) dephosphorylation over the negative control, H7 (10 µM; n = 19). HA100 treatment resulted in significant cortical granule exocytosis when evaluated by laser confocal microscopy. In addition, protein kinase assays revealed lower myosin light chain kinase activity in electroactivated oocytes (p < 0.05) and protein kinase inhibitor-treated oocytes (p < 0.05) than in negative controls, nonelectroactivated oocytes, and H7 (10 µM)-treated oocytes. Treatment with HA100 (250 µM) resulted in pronuclear formation, dephosphorylation of the 25-kDa protein, and some release of cortical granules. These observations suggest that inhibition of myosin light chain kinase, protein kinase A, and protein kinase C results in activation of porcine oocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In most vertebrates, fertilization results in the meiotic resumption of metaphase II (MII) arrested oocytes [1, 2]. The term given to this meiotic event is oocyte activation.

At oocyte activation, a Ca2+ transient activates calmodulin, which activates calmodulin-dependent protein kinase II (CaMKII). Active CaMKII triggers the ubiquitin-dependent cyclin destruction and calpain-dependent Mos destruction, thus destroying maturation promotion factor (MPF) and cytostatic factor (CSF), respectively [3–5]. Calmodulin is maintained in an inactive form by a calmodulin-binding pseudosubstrate (791–814) from myosin light chain kinase (MLCK) [6, 7]. Inhibition of MLCK activity has been shown to result in the release of the cyclin degradation machinery [3]. This observation prompted us to evaluate inhibition of MLCK in pig oocytes. Additionally, in calcium-dependent systems, phosphorylation of MLCK substrate is catalyzed by calmodulin-dependent MLCK and protein kinase C (PKC). MLCK is phosphorylated by PKC and protein kinase A (PKA) [8, 9]. This further prompted us to evaluate inhibition of other protein kinases including PKC and PKA.

In the pig, oocyte activation at fertilization is accompanied by an increase in intracellular free calcium ([Ca2+]i) in the oocyte [10, 11], pronuclear development, cortical granule (CG) exocytosis, and dephosphorylation of a 25-kDa protein to a 22-kDa protein [12]. Typically, activation is initiated when the sperm fuses with the oocyte. Parthenogenetic activation can also be induced artificially by electroactivation [10], protein synthesis inhibition [13], or broad-spectrum protein kinase inhibition [14–16].

Previous studies show that short-term exposure of in vitro-matured, MII-arrested porcine oocytes to 20 µM staurosporine, a potent inhibitor of PKC, or to 2.0 mM H7, a broad-spectrum protein kinase inhibitor (PKI), could induce 91.6% and 68.1% pronuclear development, respectively [14, 15]. Long-term exposure for 24 h to a low concentration of 100 µM H7 has also been shown to induce 77.1% pronuclear development. Additionally, long-term exposure to 100 µM H7 results in the dephosphorylation of the 25-kDa protein but does not result in significant cortical granule exocytosis as measured by transmission electron microscopy (TEM) [16].

The objective of the following experiments was to determine which specific kinases must be inhibited in porcine oocytes to induce pronuclear development and dephosphorylation of the 25-kDa protein and to determine whether protein kinase inhibition could induce CG exocytosis. Additional objectives were to determine the minimum exposure to PKI to induce pronuclear development and to measure the MLCK activity in MII arrested oocytes, electroactivated oocytes, and PKI-treated oocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents

All chemicals used in medium preparation were purchased from Sigma Chemical Company (St. Louis, MO). All chemicals and reagents for SDS-PAGE and MLCK assay were purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise stated. The peptide used in the MLCK assay was synthesized by the University of Missouri-Columbia protein core facility. The PKIs were purchased from Calbiochem (La Jolla, CA).

PKIs

PKI stocks were resuspended in sterile water or dimethyl sulfoxide (DMSO) as specified by Calbiochem (Table 1) [17–34]. The presence of low levels of DMSO in culture medium had no effect on porcine oocyte activation in previous studies [35, 36]. The PKIs used in these experiments included HA100, KT5926, Iso-H7, KT5720, KT5823, KN93, and Wortmannin. Concentrations and combinations of PKIs are in Table 1. Aliquots from PKI stocks were then diluted in modified Whitten's medium to the appropriate concentration and stored at -20°C until use. At the time of oocyte treatment, aliquots were thawed and placed into wells of a Nunc four-well plate (Nunclon, Roskilde, Denmark). The culture plates were then placed in a 39°C incubator with 5% CO2 in a water-saturated air atmosphere and equilibrated for 1.5 h. After equilibration, 25 cumulus-free oocytes (CFO) were placed into each well. Additionally, because the PKIs KT5720, KT5823, KT5926, and KN93 are light sensitive, all manipulations were done with minimal light exposure.


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  		TABLE 1. Concentrations and combinations of PKIs, the kinases that should be inhibited, and the diluent specified by Calbiochem.

Controls

The positive control for pronuclear development was 100 µM H7, which has been shown to induce pronuclear development in 77.1% of oocytes after a 24-h exposure [16]. Electroactivation of oocytes by standard conditions [15] has been shown to induce dephosphorylation in 100% of oocytes and CG exocytosis in 100% of oocytes; therefore, electroactivated oocytes were chosen for the positive control for protein profile analysis and CG exocytosis. After electroactivation, oocytes were transferred to HEPES-buffered Tyrode's medium containing 0.1% polyvinylalcohol (PVA-HbT [37]) for a 6-h development period before assessment; PVA-HbT represents a BSA-free medium. H7 (10 µM) does not effect pronuclear development, protein dephosphorylation, or CG exocytosis and served as the negative control for these experiments. A second negative control was used in the laser confocal microscopic assessment of CG exocytosis and in experiment one of the MLCK assay, which consisted of CFO that did not receive an electric shock but that were cultured for 6 h in PVA-HbT like those that were electroactivated.

In Vitro Oocyte Maturation

Ovaries from prepubertal gilts were collected at the local abattoir and transported to the laboratory at 25°C in physiological saline containing penicillin G (20 IU/ml), streptomycin (20 µg/ml), and gentamicin (25 µg/ml). Antral follicles with a diameter of 3–6 mm were aspirated with a 16-gauge needle and a syringe. The follicular fluid was pooled in a 50-ml conical centrifuge tube. After a 5-min sedimentation, the sediment was collected, filtered, and diluted in PVA-HbT.

Cumulus-oocyte complexes (COC) with intact, unexpanded cumulus cells and uniform cytoplasm were selected from the follicular debris, placed in PVA-HbT, and washed twice through fresh medium. Ten COC were transferred to each sterile 50-µl drop of BSA-free Whitten's medium supplemented with 10% porcine follicular fluid, 10 IU/ml eCG, and 10 IU/ml hCG and cultured for 20–22 h under CO2 equilibrated paraffin oil. COC were then rinsed twice in PVA-HbT, transferred to fresh medium without hormones, and cultured in the same conditions for an additional 22–24 h. Culture conditions consisted of a temperature of 39°C and a water-saturated atmosphere of 5% CO2 in air.

After 42–44 total hours of maturation, COC were stripped of cumulus cells by vortexing for 7 min in 0.3 M mannitol containing 0.3 mg/ml hyaluronidase. Oocytes with homogenous cytoplasm and an intact plasma membrane were chosen for activation by protein kinase inhibition.

In Vitro Maturation Rates

To evaluate in vitro maturation rates, untreated oocytes were evaluated for meiotic phase (ranging from germinal vesicle to MII, or pronuclear). Oocytes were mounted on glass slides, fixed, and stained with filtered 1% (w:v) aceto-orcein by a procedure previously published [15]. After staining, oocytes were evaluated using a Nikon Diaphot 300 microscope (Nikon, Garden City, NY) equipped with Hoffman Modulation Contrast Optics (Greenvale, NY) at a magnification of x400 [38].

Pronuclear Development Induced by Protein Kinase Inhibition

In a series of six experiments, oocytes were cultured in modified Whitten's medium containing various concentrations of PKI for 24 h (replications = 3–5; Table 1). After treatment, oocytes were fixed by the same method described above.

Minimum Exposure of PKI to Induce Pronuclear Development

Once it was determined which concentrations and combinations of PKI could induce pronuclear development, the minimum exposure of these compounds was evaluated. CFO were cultured in Whitten's medium containing HA100 (250 µM) or the combination mixture of WKI (1 µM Wortmannin, 75 nM KT5720, and 50 µM Iso-H7) for 6, 12, 18, or 24 h. After treatment, CFO were removed from PKI culture and rinsed three times in PVA-HbT to remove PKI and then cultured in PVA-HbT for 6 h to allow pronuclear development. Cultured CFO were mounted, fixed, stained, and evaluated for chromosomal configuration as described above.

Evaluation of Protein Profiles for the Appearance of the 22-kDa Protein

To indirectly determine which PKI could induce the dephosphorylation of the 25-kDa protein to the 22-kDa protein that is normally seen after fertilization [12], protein profiles of radiolabeled oocytes were evaluated. After treatment with HA100 for 12 h or Wortmannin, KT5720, Iso-H7, or the combination mixture of these three inhibitors for 24 h, CFO were immediately labeled for 4 h in a 25-µl drop of Whitten's medium containing 1.15 mCi L-[35S]methionine/ml (New England Nuclear [NEN] Corp., Boston, MA). After labeling, CFO were rinsed seven times through successive 50-µl drops of L-[35S]methionine-free Whitten's medium. BSA was removed by rinsing CFO in a 50-µl drop of physiological saline and then rinsing in 2 ml of PVA-HbT. Oocytes were placed individually into 0.5-ml microcentrifuge tubes with 15 µl of SDS lysis buffer and stored at -80°C.

Protein profiles were analyzed by using one-dimensional SDS-PAGE and fluorography [15].

Laser Confocal Microscopic Assessment of CGs

Protein kinase inhibitors that could induce pronuclear development and dephosphorylation of the 25-kDa protein were evaluated for their effectiveness in inducing CG exocytosis. Treatment groups included HA100 (250 µM) and the combination mixture of WKI: both of these treatments should inhibit MLCK, PKA, and PKC (Table 1). The length of PKI exposure was based on the minimum exposure times evaluated above.

CG evaluations were based on a previous report [39]. Briefly, after treatment, zonae pellucidae were removed by placing CFO in PBS containing 0.1% (w:v) pronase. Oocytes were then washed three times in PBS before fixing at room temperature with 3.7% (w:v) paraformaldehyde in PBS for at least 30 min. Oocytes were again washed three times in PBS containing 3 mg/ml BSA and 300 mM glycine for a total wash time of 15 min. Oocytes were treated for 5 min in PBS containing 0.1% (v:v) Triton X-100 and cultured in 100 µg/ml fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) PBS for 30 min. After treatment, oocytes were mounted on slides and observed by laser confocal microscopy. The confocal microscopy was performed using a Bio-Rad (Richmond, CA) MRC-600 equipped with a krypton-argon ion laser mounted on an Optiphot II Nikon microscope equipped with a x60 objective (Nikon). The CGs visible at the cortex were counted on a square of 100 or 1000 µm2 (depending on density), and the average number of CGs/100 µm2 of cortex in each oocyte was calculated.

TEM Assessment of CGs

Treated CFO were processed and fixed similarly to those in a previously published report by Mayes et al. [15]. After treatment, oocytes were further processed by postfixing with buffered 1% osmium tetraoxide for 1 h, stained with 1% aqueous uranyl acetate, dehydrated in a graded ethanol series, and embedded in epoxy resin. Ultrathin sections were cut with a diamond knife and poststained for 20 min with uranyl acetate. Ultrathin sections were then stained for 3 min in lead citrate. Sections were analyzed in a Hitachi H-600 transmission electron microscope (Missei Sangyo America Ltd., Mountain View, CA).

MLCK Activity of Oocytes

In the first MLCK assay, MLCK activity was compared in electroactivated and nonelectroactivated oocytes by using a MLCK assay [40–44] that was modified to measure activity in oocytes. This assay was done to establish standard MLCK levels in in vitro porcine MII oocytes and electroactivated oocytes. Oocytes were electroactivated and cultured for 6 h in PVA-HbT. Nonelectroactivated MII arrested oocytes were also cultured for 6 h in PVA-HbT. After the 6-h culture, oocytes were lysed in groups of 25 in 20 µl lysis buffer (25 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, and 0.1% [v:v] Tween 80). Oocyte samples were then frozen at -80°C. To further lyse the oocytes, the lysate tubes were frozen and thawed twice immediately before the initiation of the assay. Preliminary data did not show a difference in activity per oocyte between lysates of 25, 50, and 75 oocytes; therefore 25 oocytes per lysate were chosen for analysis.

The reaction consisted of a 100-µl volume of 40 mM HEPES, pH 7.0, 5 mM Mg acetate, 0.1 mM EGTA, 0.55 mM CaCl2, 1 mg BSA/ml, 0.1% (v:v) Tween 80, 1 µM okadeic acid, and a 0.5 mM synthetic peptide based on the sequence of MLCK substrate (791–814) NH3-SFYNSTARQPRKK-COOH [41]. The reaction buffer stocks were then stored at -20°C. Immediately before the assay, 5 µM [{gamma}-32P]ATP (30 Ci/mmol; NEN) was added to the reaction buffer, mixed, and then transferred to the 20-µl oocyte lysate, resulting in a total reaction volume of 120 µl.

The incubations progressed at 30°C for 0–6 min to establish linearity within the treatment group. At each 1-min time point, the reaction tubes were gently vortexed, and 15 µl of the reaction was removed and applied to 2.5 x 1.5-cm phosphocellulose ion-exchange P81 filter paper (Whatman Inc., Fairfield, NJ). The filter papers were successively washed for 5 min in 75 mM phosphoric acid for a total of five washes [44]. Additionally, three reactions were carried out simultaneously during each assay; electroactivated oocytes, nonelectroactivated oocytes, and a background control. The background control contained 20 µl of lysis buffer without oocytes, and the 100-µl reaction buffer.

After washes, filter papers were air-dried for 2.5–3.0 h and placed in scintillation vials with 5 ml of Scintiverse BD (Fisher Scientific). After scintillation counting, linearity within the assay was determined.

In the second assay, MLCK activity was measured in PKI-treated CFO and compared to the MCLK activity of the negative control (10 µM H7). CFO were cultured in Whitten's medium containing WKI for the minimum exposure period. After WKI exposure, oocytes were lysed in groups of 25 as described above. The MLCK assay was also performed as above except for the addition of the appropriate concentration of inhibitors to the reaction buffer. In this experiment, three reactions were carried out simultaneously during each assay; WKI-treated oocytes, 10 µM H7-treated oocytes (negative control), and a background control. After counting, linearity within the assay was determined, and the MLCK activity of WKI-treated oocyte was compared to the negative controls (10 µM H7).

Statistical Analysis

Statistical analysis was performed on pronuclear development and protein profile data by constructing confidence intervals about the mean percentage data using the normal approximation for a binomial proportion [45]. Mean CG densities and MLCK activity were evaluated using the multivariate general linear hypothesis (MGLH) on the SYSTAT program [46]. In all experiments, p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Oocyte Maturation Rates

In vitro maturation rates were measured in 3 replications. More than 90% of oocytes reached MII in all 3 replications.

Pronuclear Development Induced by Protein Kinase Inhibition

In a series of six experiments, oocytes were evaluated for pronuclear development after treatment with specific PKI. In experiment one, HA100 (250 and 350 µM) treatment resulted in 74.0% and 73.5% pronuclear development, which was significantly (p < 0.05) higher than that in negative controls (10 µM H7; Fig. 1A). In the second experiment, the effect of MLCK and CaMKII inhibitor KT5926 on pronuclear development was evaluated. KT5926 (9 nM, 18 nM, 36 nM) resulted in a level of pronuclear development of less than 29.5% and was not different from that in the negative control (10 µM H7; p > 0.05; Fig. 1B). In experiment three, oocytes were treated with KT5720 (56 nM), Iso-H7 (50 µM), or KT5823 (234 nM). These treatments did not result in significant (p > 0.05) pronuclear development (15.8%, 13.0%, and 12.3%, respectively) over the negative control (10 µM H7; Fig. 1C). KN93 (100 nM, 400 nM, 900 nM), a CaMKII inhibitor, was evaluated in experiment four and resulted in pronuclear development rates (< 8.7%) that were not different from those of negative controls (p > 0.05; Fig. 1D). In experiment five, oocytes were treated with various concentrations of Wortmannin (5 nM, 50 nM, and 500 nM). Low concentrations of Wortmannin resulted in pronuclear development rates of 7.7%, 9.1%, and 4.5%, respectively, which were lower than that of the positive control (p < 0.05; Fig. 1E). In experiment six, oocytes were treated with KT5720, Iso-H7, or Wortmannin, or a combination mixture of these inhibitors and evaluated for pronuclear development. Wortmannin (1 µM) and the combination mixture of WKI resulted in pronuclear development of 18.0% and 34.8%, respectively (Fig. 1F), which was significantly higher than that in the negative control (10 µM H7; p < 0.05).



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  		FIG. 1. Percentage of pronuclear development (± 95% confidence interval) of oocytes exposed to various concentrations of PKIs. Each experiment contains 3–5 replications. A) HA100. B) KT5926. C) Iso-H7, KT5720, or KT5823. D) KN93. (E) Wortmannin. F) Wortmannin (W), KT2720 (K), or Iso-H7 (I), or a combination of KI or WKI. Values with different letters are significantly different; abcdp < 0.05.

Minimum Exposure of PKI to Induce Pronuclear Development

Oocytes were exposed to either HA100 (250 µM) or a combination mixture of WKI for 6, 12, 18, or 24 h. After exposure, oocytes were cultured in PKI-free PVA-HbT for 6 h. The minimum exposure to HA100 was 12 h and resulted in 54.1% pronuclear development (Fig. 2). The minimum exposure to WKI was 24 h and resulted in 38.2% pronuclear development (Fig. 3).



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  		FIG. 2. Minimum exposure to 250 µM HA100 required to induce pronuclear development, after a 6-, 12-, 18-, or 24-h exposure. Values with different letters are significantly different; abp < 0.05.



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  		FIG. 3. Minimum exposure to the combination of 1 µM Wortmannin, 75 nM KT5720, and 50 µM Iso-H7 required to induce pronuclear development after a 6-, 12-, 18-, or 24-h exposure. Values with different letters are significantly different; abcdp < 0.05.

Evaluation of Protein Profiles for the Appearance of the 22-kDa Protein

After fertilization has occurred in pig oocytes, a 25-kDa protein is dephosphorylated which then appears as a 22-kDa protein as analyzed by autoradiography [12]. Appearance of this 22-kDa protein was used as a measure of activation. Oocytes with incomplete dephosphorylation showed both bands and were also included in the percentage appearance of the 22-kDa protein calculations. Only electroactivated, H7 (100 µM)-treated, and HA100 (250 µM)-treated oocytes exhibited a significant appearance (p < 0.05) of the 22-kDa protein (Table 2, Fig. 4; the percentage of oocytes that had the 22-kDa band were 78.6%, 61.1%, and 80.0%, respectively [p < 0.05]). The negative control H7 (10 µM) showed 10.5% appearance of the 22-kDa protein. The WKI combination resulted in 31.6% appearance of the 22-kDa protein but this was not significant over the negative control (p > 0.05). Additionally, inhibition by Wortmannin, KT5720, and Iso-H7 individually did not result in significant (p > 0.05) appearance of the 22-kDa protein, with rates of 29.4%, 18.8%, and 15.8%, respectively, compared to those of negative controls (Table 2, Fig. 4).


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  		TABLE 2. The percentage of appearance of the 22-kDa band after treatment with PKI.



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  		FIG. 4. L-[35S]Methionine-labeled proteins from PKI-treated oocytes. +, Positive control (electroactivated); -, negative control (10 µM H7); MLCK: treated with 1 µM Wortmannin; PKA: treated with 75 nM KT5720; PKC, treated with 50 µM Iso-H7, All 3: treated with 250 µM HA100. Arrows denote the 25-kDa and 22-kDa bands.

Laser Confocal Microscopic Assessment of CG Exocytosis

CG exocytosis was also used as a measure of activation in this experiment. After treatment with HA100 for 12 h or the combination of WKI for 24 h, FITC-PNA-labeled CFO were evaluated by laser confocal microscopy. The average number of CGs/100 µm2 of cortex was determined for each treatment. There were 3 replicates.

Electroactivated (positive control) oocytes had a significant loss of CGs as shown by the lowest (p < 0.001) CG density of 15.7 ± 2.9 CGs/100 µm2 of cortex (Fig. 5B). Both negative controls (6 h PVA-HbT and H7 [10 µM]) had the highest (p < 0.001) mean CG densities, of 53.3 ± 3.3 and 54.6 ± 3.0, respectively (Fig. 5, A and E). HA100- and WKI-treated oocytes had CG mean densities of 39.0 ± 2.7 and 37.4 ± 2.9 CGs/100 µm2 of cortex, respectively. These reduced densities were significantly lower than those of negative controls and significantly higher than those of positive controls (p < 0.001; Fig. 5, C and D).



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  		FIG. 5. Effect of a 12-h exposure to 250 µM HA100 or a 24-h exposure to WKI on CG evaluated by laser confocal microscopy. A) Negative control (6 h HbT). B) Positive control (electroactivated). C) 250 µM HA100. D) WKI. E) Negative control (10 µM H7). Bar = 20 µm.

TEM Assessment of CG Exocytosis

CG exocytosis was also evaluated by TEM. After treatment, oocytes were processed for TEM and examined for the presence of CGs. Neither treatment with HA100 (n = 5), the WKI combination (n = 5), nor the negative control (n = 8) resulted in detectable CG release in the oocytes examined (Fig. 6, B–D). In the positive control, 4 of 5 oocytes had released their CGs (Fig. 6A).



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  		FIG. 6. Effect of a 12-h exposure to 250 µM HA100 or a 24-h exposure to WKI on CG exocytosis evaluated by TEM. A) Positive control (electroactivated). B) Negative control (10 µM H7). C) 250 µM HA100. D) WKI. Arrowheadss denote CGs. ZP, zona pellucida.

MLCK Activity of Oocytes

In the first MLCK assay, a standard MLCK activity was determined for electroactivated oocytes and nonelectroactivated oocytes. The difference in activity of MLCK was calculated by subtracting the number of counts in the background control sample from the number of counts in the treatment sample and establishing a ratio between these values. The counts were taken at a linear time point in each assay. The mean MLCK activity of electroactivated oocytes was 28% lower than that of nonelectroactivated oocytes (p < 0.05, n = 6).

In the second MLCK assay, MLCK assays were performed on PKI-treated oocytes and 10 µM H7-treated oocytes to determine whether MLCK activity was truly being inhibited in the oocyte lysate. Mean MLCK activity in the oocyte lysate containing PKIs was 38% lower than that in the negative control (10 µM H7; p < 0.05, n = 4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Fertilization provides a signal that activates the cell cycle machinery, resulting in uninterrupted divisions of the embryo. It is unclear whether this signal is via a sperm-receptor-mediated pathway [47–53] or from a compound contained in the sperm, which enters the oocyte [54]. However, it is known that maturation promotion factor (MPF) and cytostatic factor (CSF) maintain the MII arrest. At oocyte activation, a Ca2+ transient activates calmodulin, which activates CaMKII. CaMKII activates ubiquitin-dependent cyclin destruction and calpain-dependent Mos degradation, thus destroying both MPF and CSF activity, respectively [3–5]. A calmodulin-binding peptide from MLCK, MLCK (791–814), prevents exogenous Ca2+ from triggering cyclin degradation and maintaining MII arrest [6, 7, 55]. Inhibition of MLCK activity has been shown to result in the release of the cyclin degradation machinery [3]. This observation prompted us to evaluate inhibition of MLCK activity in pig oocytes.

This series of experiments was conducted to determine which kinase(s) may be involved in activation of porcine oocytes. Once it was determined which kinases must be inhibited to induce pronuclear development, then the effect of inhibiting these specific protein kinases on other measures of activation was determined. The measures of activation used in these experiments include pronuclear development, dephosphorylation of the 25-kDa protein to a 22-kDa protein, and CG exocytosis. The minimum exposure to these PKIs required to induce pronuclear development was also measured. Since MLCK activity was involved in activation, an enzymatic assay for this kinase was developed to measure MLCK activity of oocytes.

In a series of six experiments, oocytes were cultured in various PKIs, and the effect on pronuclear development was determined. Inhibition by Wortmannin (1 µM), which should inhibit MLCK (Table 1), resulted in significant pronuclear development (18.0%). However, inhibition by HA100 and a combination of WKI resulted in a much higher induction of pronuclear development (74.0% and 34.8%, respectively; p < 0.05). These treatments should inhibit MLCK, PKA, and PKC (Table 1). Preliminary experiments using lower concentrations of WKI did not result in significant pronuclear development. Treatments that should inhibit PKA, PKC, PKG, and CaMKII, individually, or a combination of PKA and PKC, did not result in significant pronuclear development. Once it was established that inhibition by HA100 and WKI resulted in efficient induction of pronuclear development, the minimum exposure to these inhibitors was determined to be 12 h and 24 h, respectively.

Treatment with the MLCK, PKA, and PKC inhibitor HA100 also resulted in the dephosphorylation of the 25-kDa protein to a 22-kDa protein in 80.0% of oocytes tested. Inhibition of these kinases by WKI did result in a 31.6% appearance of the 22-kDa band, but it was not significantly higher than results in negative controls. Ding et al. [12] showed that this dephosphorylation event occurs at the same time as pronuclei formation. Consistent with these data, the percentage of pronuclear formation in response to WKI in experiment 6 (Fig. 1F) is similar to the percentage of appearance of the 22-kDa protein (Table 2).

Treatment with HA100 and WKI induced CG exocytosis that was significantly higher than that in negative controls, yet significantly lower than in positive controls as evaluated by laser confocal microscopy. There was not significant CG exocytosis as evaluated by TEM. Prather et al. [16] showed that the broad-spectrum PKI H7 (100 µM) also did not induce CG exocytosis when evaluated by TEM. Wang et al. [56] showed that inhibition by 3 µM staurosporine for 30 min, which should inhibit PKA, PKC, and PKG, resulted in very little CG release. Additionally, Wang et al. [56] evaluated high concentrations with short exposure to staurosporine. In our experiment, we concentrated on physiologically relevant levels of PKI for longer exposure periods.

On the basis of the above data, activation of pig oocytes appears to be dependent on MLCK inhibition in combination with PKA and PKC inhibition; therefore, it was also important to establish standard MLCK levels in MII and electroactivated oocytes and MLCK activity in PKI-treated oocytes. As expected, the percentage of MLCK activity in electroactivated oocytes was lower than in MII arrested oocytes. This indicates that the artificial Ca2+ burst during electroactivation may overpower the cell cycle hold from the calmodulin-binding peptide of MLCK and trigger cyclin degradation and cell cycle progression. The decrease in MLCK activity in the presence of MLCK, PKA, and PKC inhibitors shows that MLCK is truly inhibited in the oocyte lysate. Thus, the concentration of WKI (1 µM, 75 nM, and 50 µM, respectively) appears to be adequate in inhibiting MLCK in oocytes.

In conclusion, inhibition of MLCK, PKA, and PKC by HA100 results in activation of porcine oocytes as measured by pronuclear development and dephosphorylation of the 25-kDa protein. Inhibition of MLCK, PKA, and PKC by a combination of WKI, and inhibition of MLCK alone by Wortmannin (1 µM) also results in low levels of pronuclear development. As expected, MLCK activity is lower in oocytes artificially activated by an electrical stimulus or by MLCK, PKA, and PKC inhibition. The MLCK assay can be used as an important tool to investigate MLCK activity in in vivo-derived fertilized oocytes compared to in vivo-derived MII arrested oocytes. These experiments help elucidate some of the mechanisms responsible for porcine oocyte activation.


   ACKNOWLEDGMENTS
 
The authors would like to acknowledge Ryan Cabot and Tom Cantley for traveling to the abattoir to collect ovaries. We would also like to thank Jonathan Green for technical assistance on the MLCK assay.


   FOOTNOTES
 
1 This work was funded in part by Food for the 21st Century. This manuscript is a contribution from the Missouri Agricultural Experiment Station Journal series number 12,679. Back

2 Correspondence: Randall S. Prather, 162 Animal Science Research Center, University of Missouri-Columbia, Columbia, MO 65211. FAX: 573 882 6827; pratherr{at}missouri.edu Back

Accepted: February 17, 1999.

Received: October 17, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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