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Biology of Reproduction 62, 292-302 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Butyrolactone I Reversibly Inhibits Meiotic Maturation of Bovine Oocytes,Without Influencing Chromosome Condensation Activity1

Michal Kubelkaa, Jan Motlíka, Richard M. Schultz2,b, and Antonín Pavloka

a Institute of Animal Physiology and Genetics, 277 21 Libechov, Czech Republic b Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle (GV) breakdown (GVBD) in bovine oocytes in a concentration-dependent manner; GVBD was almost totally inhibited over the course of 24–48 h of culture when 100 µM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34cdc2 kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 µM BL I did not inhibit MAP kinase activity present in extracts obtained from metaphase II-arrested bovine eggs that possess high levels of MAP kinase activity. Nevertheless, the formation of highly condensed bivalents was observed in 78% of the BL I-treated GV-intact oocytes. This result suggests that chromosome condensation during first meiosis in bovine oocytes does not require the activity of either p34cdc2 kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34cdc2 kinase or MAP kinase, or inducement of GVBD. The BL I-induced block of GVBD for 24 h was reversible, and a subsequent 24-h culture resulted in 90% of oocytes reaching metaphase II with emission of the first polar body. Correlated with the progression to and arrest at metaphase II was the full activation of both p34cdc2 and MAP kinases. The reversibility after 48 h of culture in BL I was partially decreased when compared to that achieved after an initial 24-h culture. Fertilization in vitro of these eggs resulted in a high incidence of both sperm penetration and pronucleus formation (88% and 70%, respectively).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian oocytes, including cattle oocytes, are arrested in vivo at the diplotene (dictyate) stage of first meiotic prophase, the so-called germinal vesicle (GV) stage. When fully grown oocytes are removed from their follicles, they can resume meiosis spontaneously if cultured in vitro in a suitable culture medium. During meiotic maturation, major morphological changes occur, including germinal vesicle breakdown (GVBD), chromosome condensation, and rearrangement of microtubule network, all of which are necessary for reaching the first meiotic metaphase (MI). Oocytes then proceed directly through anaphase I and telophase I without any detectable decondensation of the chromosomes to reach the second meiotic metaphase (MII), at which point they arrest. Resumption and completion of the second meiotic division occurs after either fertilization or parthenogenetic activation.

The major morphological changes that occur during oocyte maturation in several species are accompanied by major changes in protein phosphorylation (Xenopus [1], starfish [2], mice [3,4], sheep [5], goats [6], and cattle [7]). Correlated with this burst of phosphorylation is the activation of two major M-phase kinases, namely, p34cdc2 protein kinase and mitogen-activated protein (MAP) kinase. p34cdc2 protein kinase (known also as cdc2 kinase or cdk1 kinase) is the catalytic subunit of a complex known as MPF (M-phase promoting factor), described earlier by Masui and Markert [8], and believed to be the key factor that regulates the G2/M transition, not only in meiotic cells (oocytes) but also in somatic cells. The activation of MPF occurs at two levels. The first is association of p34cdc2 with the regulatory subunit, cyclin B, which is already synthesized and accumulated during interphase [9]. The second level is the phosphorylation and dephosphorylation of different sites on p34cdc2 itself [10–14].

MAP kinase, a serine/threonine kinase, belongs to a family of MAP kinases. MAP kinases are also termed extracellular signal-regulated kinases (ERKs), because they are important intermediates in the signaling pathway by which an external stimulus at the cell surface effects changes in the cell [15]. The activation of MAP kinases requires phosphorylation on both tyrosine and threonine residues by an upstream kinase identified as a dual specific MAP kinase kinase (MAPKK), also termed MEK [16,17]. Apart from the numerous somatic cell types, in which MAP kinase is activated in response to growth factors, the activation of MAP kinase occurs at the onset of oocyte maturation in number of species including Xenopus [18–20], starfish [21,22], and also mammals, such as mice [23–25], cattle [26], goats [27], or pigs [28].

MPF (or p34cdc2 kinase) appears to regulate directly some of the morphological changes that occur during M phase. For example, MPF can convert the interphase status of microtubules towards a metaphase configuration in vitro [29]. p34cdc2 also acts directly as a lamin kinase, phosphorylating lamins on mitotic phosphorylation sites and causing nuclear lamina disassembly [30]; the disassembly of nuclear lamina, in turn, is an essential prerequisite for the process of nuclear envelope breakdown [31,32]. The correlation between chromosome condensation during M phase and MPF/p34cdc2 kinase activation is less clear. The process of chromatin condensation during M phase in many species is accompanied by phosphorylation of histones H1 and H3. Although H1 serves as an excellent substrate in vitro for p34cdc2, it is not clear whether histone H1 is phosphorylated by p34cdc2 in vivo and what the role is (if any) of histone H1 phosphorylation in chromosome condensation (for review see [33]). Furthermore, the p34cdc2 kinase activity is not always correlated with the morphological changes during meiotic maturation. For example, during the transition between MI and MII, the chromosomes remain condensed and microtubules stay in a metaphase state, yet p34cdc2 kinase activity drops abruptly during anaphase I and telophase I before it rises to its maximum level in MII [34,35].

The observation that activity of MAP kinase, which becomes activated in oocytes before or concomitantly with p34cdc2 kinase, remains high during the anaphase I/telophase I transition suggests that MAP kinase regulates the metaphase state of microtubules and chromosomes during this period [24,35]. Consistent with this proposal are the observations that MAP kinase is involved in changes of microtubule dynamics during the interphase-metaphase transition in Xenopus oocyte extracts [36] and that MAP kinase is associated with microtubule organizing centers involved in spindle assembly in the mouse oocytes [35].

Butyrolactone I (BL I), which acts as a competitive inhibitor of ATP, is a potent and specific inhibitor of cyclin-dependent kinases (cdks) and has few inhibitory effects on other protein kinases such as MAP kinase [37,38]. We used BL I as a tool to distinguish the roles of MAP kinase and p34cdc2 kinase during the maturation of bovine oocytes. During the course of these studies, we noted that BL I reversibly inhibits GVBD, and we capitalized on this by developing a two-step culture system to provide sufficient time for oocytes to develop full meiotic competence, which in turn would foster a better starting point to achieve successful cytoplasmic maturation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

The following chemicals were used: BL I was from Banyu Pharmaceutical Co. (Tsukuba, Japan); histone H1 was from Boehringer (Mannheim, Germany); crystallized BSA, silicone oil, sodium-pyruvate, calcium-lactate, and Coomassie Blue were from Serva (Heidelberg, Germany); tissue culture medium 199 (TCM 199; 10-strength concentration stock) and NaHCO3 (7.5% stock) were from Sevac (Prague, Czech Republic); bovine serum (inactivated; BOS) was from Bioveta (Ivanovice, Czech Republic); porcine FSH (radioiodination grade) was from Biogenesis (Poole, UK); Suigonan PG-600 (FSH/LH gonadotropins) was from Intervet, International B.V. (Boxmeer, Holland); [{gamma}-32P]ATP was from Amersham (London, UK); myelin basic protein (MBP), polyvinyl alcohol (Mr = 30 000–50 000), and all other reagents were from Sigma (St. Louis, MO). Deionized and Nanopure (Barnstead/Thermolyne, Dubuque, IA) filtered water was used for all media. The plastic dishes were from Nunclon (Roskilde, Denmark).

Oocyte Collection and Culture

Ovaries, collected from slaughtered cows, were transported in physiological saline at 20°C to the laboratory. The ovaries were briefly washed for 20 sec in 70% ethanol and then twice in physiological saline. Follicles 2.5- to 8-mm in diameter were dissected with fine scissors, placed into 90-mm Petri dishes with basic culture medium (see below), and punctured and pressed with a bent preparation needle to isolate the oocytes. For oocyte culture in vitro, only healthy-looking oocyte-cumulus cell complexes (OCC) were selected [39]. The OCCs were washed twice in basic culture medium and transferred into 4-well Nunclon dishes with 0.5 ml medium under silicone oil. The OCCs were cultured either in follicular fluid (see below) or in basic culture medium.

Follicular fluid was isolated from follicles 2.5–5 mm in diameter. The follicles were washed with PBS, and then individual follicles were punctured and pressed with a bent preparation needle to isolate the follicular fluid that also contained the oocyte. The oocytes were removed, and the remaining follicular fluid, which contained tissue debris, was centrifuged. The osmolality of the supernatant was then measured and adjusted to 300 mOsm with water and supplemented with 50 IU/ml penicillin K salt, 50 IU/ml streptomycin sulfate, and 125 ng/ml amphotericin B before use for oocyte culture.

Basic culture medium consisted of 9.4 ml of a 10-strength stock solution of TCM 199, 2.1 ml of a 7.5% solution of NaHCO3 in 100 ml of H2O supplemented with 9.5 mM HEPES, 1.82 mM sodium-pyruvate, 50 IU/ml penicillin K salt, 50 IU/ml streptomycin sulfate, and 125 ng/ml amphotericin B. This basic culture medium was used either without supplements (for manipulation) or supplemented with 3 mg/ml polyvinyl alchohol (PVA) or 3 mg/ml PVA + 100 ng FSH, or with 3 mg/ml crystallized BSA or 10% BOS.

In the experiments examining the effect of BL I and/or OA, the basic culture medium containing 3 mg/ml BSA was used, supplemented with either 2 µl/ml of dimethyl sulfoxide (DMSO) or 6, 12, 25, 50, 100, or 200 µM of BL I, or with 100 µM BL I and 2.5 µM OA. BL I was prepared as 50 mM stock solution in DMSO; OA was prepared as 50 mM stock in DMSO.

In the experiments assessing the reversibility of BL I action on oocyte maturation, the oocytes were cultured either in follicular fluid or in basic culture medium supplemented with the following compounds: 3 mg/ml PVA, 3 mg/ml BSA, 3 mg/ml PVA + 100 ng FSH, or 10% BOS. All these media served as controls or they were supplemented with 100 µM BL I to prepare meiosis-inhibiting media.

In the reversibility experiments, after culture for either 24 or 48 h in either control medium or meiosis-inhibiting medium, one half of the oocytes were used to assess morphological changes or for biochemical assays. The other half were transferred to maturation medium for second-step culture. In the second step of oocyte culture (maturation), both groups of oocytes (control and experimental) were washed three times in basic culture medium before they were transferred to maturation medium composed of TCM 199 (8.5 ml of a 10-strength stock solution, 3.8 ml of a 7.5% solution of NaHCO3 in 100 ml H2O, supplemented with 1.82 mM sodium-pyruvate, 50 IU/ml penicillin K salt, 50 IU/ml streptomycin sulfate, 125 ng/ml amphotericin B, 10 IU/ml gonadotropins [PG-600], and 10% BOS).

All cultures were done in a humidified atmosphere composed of 5% CO2, 10% O2, and 85% N2 at 39°C.

Sperm Preparation and Fertilization

For sperm preparation and fertilization, the following fertilization medium was used: 115 mM NaCl, 5.35 mM KCl, 0.52 mM KH2PO4, 0.40 mM MgSO4, 44.64 mM NaHCO3, 2.27 mM calcium-lactate, 1.82 mM sodium-pyruvate, 6.29 mM HEPES acid, 50 IU/ml penicillin K salt, 50 IU/ml streptomycin sulfate, and 125 ng/ml amphotericin B, supplemented with 10% BOS. Osmolality of fertilization medium was 300 mOsm, pH 7.7.

Two frozen sperm pellets obtained from one bull were thawed by suspending each pellet in 1.5 ml of fertilization medium at 39°C. The two samples were then pooled and centrifuged at 350 x g for 8 min, and the sperm pellets were washed twice in fertilization medium. After a second centrifugation, the pelleted sperm were incubated in fertilization medium for 15 min and divided into two equal parts, and each half was layered under 1 ml of fertilization medium and incubated for 15 min at 39°C for swim-up. The supernatants containing the highly motile spermatozoa were pooled and centrifuged. The sedimented spermatozoa were diluted to a final concentration of 0.5 x 106/ml in fertilization medium containing 4 IU heparin per ml and then placed into wells of four-well Nunclon dishes and incubated for 3 h at 39°C.

For fertilization, 30–40 OCCs, which had been washed three times in fertilization medium, were placed into each well containing the spermatozoa. Eighteen to 20 h after being added to spermatozoa, the oocytes were fixed to assess the extent of fertilization.

Fixation, Staining of Oocytes, and Morphological Analysis

At the end of culture, the cumulus and corona radiata of the oocytes were removed by a 2-min treatment with 2.5 µg/ml hyaluronidase followed by mechanical stripping of the cumulus cells with a manipulation pipette. The oocytes were mounted on microscope slides with vaseline, covered with a cover glass, and fixed in ethanol-acetic acid 3:1 for 24 h. Staining was performed with 2% orcein in 50% aqueous-acetic acid, 1% sodium citrate. The slides were then placed in 40% acetic acid and observed with a phase contrast NU Zeiss microscope (Jena, Germany).

Myelin Basic Protein and Histone H1 Kinase Double Assay

Activities of histone H1 and of MBP kinases, reflecting p34cdc2 and MAP kinase activities, respectively, were measured in oocyte extracts by their capacity to phosphorylate external substrates, namely histone H1 and MBP. At each time interval during the culture, 10 oocytes per sample were collected, washed four times in PBS, and transferred in 3 µl of PBS into Eppendorf tubes. Samples were immediately frozen on dry ice and stored at -80°C until assays were performed. The histone H1 and MBP kinase activities were measured according to Motlík et al. [40]. Briefly, 5 µl of buffer A (40 mM 3-[n-morpholino] propanesulfonic acid [MOPS] pH 7.2, 20 mM para-nitrophenyl phosphate, 40 mM beta-glycerophosphate, 10 mM EGTA, 0.2 mM EDTA, 2 mM dithiothreitol, 0.2 mM Na3VO4, 2 mM benzamidine, 40 µg/ml leupeptin, and 40 µg/ml aprotinin) was added to each sample, and the samples were subjected to three rounds of freezing and thawing on dry ice. After the final thawing, the tubes were briefly vortexed and centrifuged at 10 000 x g for 15 sec. The kinase reaction was initiated by addition of 5 µl of buffer B (100 mM MOPS pH 7.2, 20 mM para-nitrophenylphosphate, 40 mM beta-glycerophosphate, 20 mM MgCl2, 10 mM EGTA, 0.2 mM EDTA, 5 µM cAMP-dependent protein kinase inhibitor, 2 mM benzamidine, 40 µg/ml leupeptin and 40 µg/ml aprotinin, 600 µM ATP, 2 mg histone H1/ml, and 3 mg MBP/ml) with 500 µCi/ml [{gamma}-32P]ATP (10 mCi/ml; Amersham). The reaction was conducted for 30 min at 30°C and terminated by the addition of 10 µl double-strength concentrated SDS PAGE sample buffer and boiling for 3 min. After electrophoresis on 15% SDS PAGE gels [41], the gels were stained with Coomassie Blue R250, destained overnight, dried, and autoradiographed.

SDS PAGE and Immunoblotting

Immunoblotting with antibodies against p44 (ERK1) and p42 (ERK2), two members of the MAP kinase family, was carried out to confirm that the activity of MBP kinase reflected that of MAP kinase. At each time interval, 10 oocytes per sample were collected, washed five times in protein-free media (PBS), lysed in 10 µl double-strength SDS sample buffer containing 5% 2-mercaptoethanol, and stored immediately at -80°C until electrophoresis. Samples were separated on 9% SDS PAGE gels [41] in which the acrylamide:bisacrylamide ratio in the separation gel was 100:1. Separated proteins were transblotted to Immobilon-P (Millipore, Bedford, MA) membranes using a tank-buffer apparatus (200 mA, 1 h). Blots were incubated in 10% teleost gelatin (Sigma) dissolved in 0.05% Tween-20 in Tris-buffered saline pH 7.4 (TTBS) for 1 h before development with anti-ERK1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; sc-94, 1:1000), followed by secondary anti-rabbit, horseradish peroxidase-linked Ig (Amersham; 1:5000); the blots were incubated with each antibody for 1 h at room temperature. The blots were then washed at least 5 times for at least 10 min for each wash in TTBS and then developed with an ECL kit according to the instructions of the manufacturer (Amersham).

Statistical Analyses

Instat software was used for statistical evaluation (GraphPAD Software, San Diego, CA). The effect of BL I diluted by different culture media on meiotic maturation (proportion of oocytes in GV or MII stage) was evaluated by chi square analysis with Yates (continuity) correction.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of BL I Concentration and Different Culture Media on BL I-Mediated Inhibition of Meiosis and Chromatin Configuration

Currently, the only available data on the inhibitory effect of BL I on cdks have been obtained in somatic cells, and the inhibitory effect on p34cdc2 kinase activity was observed between 20 and 50 µM [38]. Given the role of p34cdc2 in oocyte maturation, we explored the effect of BL I on bovine oocyte maturation. BL I inhibited oocyte maturation in a concentration-dependent manner when GVBD was assessed after 24 h of culture in basic culture medium containing PVA (Fig. 1). Partial degeneration of GV-intact oocytes was observed at 200 µM BL I. Inclusion of BSA in the medium resulted in a moderate shifting of the concentration dependence of inhibition of GVBD to the right, while inclusion of BOS had a very pronounced effect and, moreover, resulted in only a partial inhibition of GVBD at 200 µM BL I (Fig. 1). This loss of inhibition most likely reflected the binding of BL I to BSA or BOS components and hence reducing the effective concentration of BL I in solution.



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  		FIG. 1. Inhibition of meiotic maturation of bovine oocytes with BL I: dose-effect of BL I (at concentrations shown) in basic culture medium containing 3 mg/ml PVA, or 3 mg/ml BSA, or 10% BOS. Results were obtained in 4 replicate experiments. A total of 215 oocytes were cultured for 24 h either in basic culture medium with PVA and 2 µl/ml DMSO or BL I in DMSO; a total of 155 oocytes were cultured for 24 h either in the basic culture medium with BSA and 2 µl/ml DMSO or with BL I in DMSO. A total of 206 oocytes were cultured either in basic culture medium with BOS and 2 µl DMSO or with BOS and BL I

We selected as culture conditions basic culture medium containing 3 mg/ml of BSA and 100 µM BL I to study further the effects of BL I on inhibition of oocyte maturation. Although these conditions inhibited 89.5% of the oocytes (108 examined) from undergoing GVBD by 24 h of culture, 86% of these GV-intact oocytes were in the so-called GV-IV stage, in which condensed chromosome bivalents were seen within an intact GV (Fig. 2B). The remaining 14% of these GV-arrested oocytes displayed no signs of chromosome condensation, a situation that was observed in oocytes analyzed just after they were released from their follicles, so called GV-I oocytes (Fig. 2A), or they were degenerated (Table 1). These results, together with data on H1 kinase and MAP kinase activities (see below), suggest that chromosome condensation during the first meiotic reduction in bovine oocytes is regulated independently on either H1 kinase or MAP kinase activation.



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  		FIG. 2. Morphology of oocytes cultured in the presence of BL I. A) Freshly isolated bovine oocyte in the GV I stage. x750. B) BL I blocked bovine oocytes in GV IV stage; condensed bivalents, however, are visible in the nucleoplasm. x750. C) When bovine oocytes were cultured for 12–14 h in control medium, they reached MI stage. A–C, x750 (published at 68%)


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  		TABLE 1. Morphology of oocytes treated with BL I alone or together with OA

Effect of BL I on Histone H1 and MAP Kinase Activities in Bovine Oocytes

The observation that chromosome condensation occurred in many of the BL I-treated, GV-intact oocytes under conditions that would probably not result in the activation of either MPF or MAP kinase (each of which is normally activated during the course of oocyte maturation), led us to examine the effect of BL I on the activity of these two protein kinases using two different assays. One assay measured the ability of p34cdc2 kinase and MAP kinase to phosphorylate histone H1 and MBP, respectively, as external substrates. Since MBP is a substrate for a number of protein kinases, the activity of MAP kinase was also assayed by immunoblotting, using an antibody that recognizes two members of MAP kinase family, ERK1 and ERK2. Both ERK1 and ERK2 are activated by phosphorylation, which results in a decrease in their electrophoretic mobility.

Oocytes were cultured in the basic culture medium containing BSA in either the absence or presence of BL I, and histone H1 kinase and MAP kinase activities were then assayed at different times (Fig. 3A). Both histone H1 kinase and MBP kinase possessed minimum activity levels at GV stage (time 0 h) and throughout next 6 h of culture, and then they both began to be activated approximately at the same time, i.e., after 8 h of culture, which was correlated with the time of GVBD. The activation of H1 kinase continued and reached the first maximum after 12 h of culture, the time of MI; then it declined partially at 16 h and reached the second maximum after 20 h of culture, when most of the oocytes had reached MII stage. On the other hand, MBP kinase activity increased gradually, reaching a maximum after 12–14 h of culture and staying at the same level for up to 24 h of culture (Fig. 3A). A similar time course of MAP kinase activation was also observed using the immunoblotting assay procedure (Fig. 3B). Not only did the addition of BL I to culture medium result in a block of GVBD (see above); but also neither H1 kinase nor MAP kinase was activated in these oocytes, even after 24 h of treatment (Fig. 3, A and B, last lane). As described later, this inhibition of both GVBD, and MPF and MAP kinase activation was reversible.



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  		FIG. 3. Histone H1 kinase and MAP kinase activities in control and BL I-treated oocytes. A) Bovine oocytes were cultured in control medium (basic culture medium with BSA) for 0–24 h (C0–C24), and at the indicated times (C1, C2, etc.), the samples were collected for histone H1 and MBP double-kinase assay by adding both substrates in a kinase cocktail. These oocytes were compared with oocytes cultured for 24 h in the presence of 100 µM BL I (BL 24). B) Changes in phosphorylation status of two MAP kinases (ERK1 and ERK2) as detected by mobility shifts in samples from control and BL I-treated oocytes shown by Western blotting. Ten oocytes were used for each sample in both assays. The experiment was conducted three times, and a representative example is shown

Given the specificity of BL I for inhibiting cdks [37,38], it was unlikely that the observed inhibition of MAP kinase activity during oocyte maturation was due to direct inhibition of this enzyme by BL I. Nevertheless, this was a formal possibility. To determine whether BL I could directly inhibit MAP kinase activity in our system, the effect of BL I on p34cdc2 kinase and MAP kinase activities in oocyte extracts prepared for MII eggs that contained elevated levels of each of these activities was measured. While low concentrations of BL I did not inhibit the activity of either protein kinase, 100 µM or 200 µM BL I totally inhibited p34cdc2 kinase activity but had little if any effect on MAP kinase activity (Fig. 4). In addition, the presence of OA, which is known to activate p34cdc2 kinase in vitro, did not overcome the inhibiting effect of BL I on p34cdc2 kinase activity in these extracts (Fig. 4).



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  		FIG. 4. In vitro assay of BL I effect on histone H1 and MBP kinase activities. To check the specificity of BL I-mediated inhibitory effect, samples were prepared from control oocytes cultured for 24 h, i.e., MII-stage oocytes (known to possess high histone H1 and MAP kinase activities). These samples were subjected to histone H1/MBP double-kinase assay, in which, apart from both substrates, BL I was added to the kinase cocktail in concentrations of 6–200 µM (as indicated). Lane C0, histone H1/MBP kinase activities in control, GV-stage oocytes; lane C24+OA/BL I, kinase activities in MII oocyte extracts incubated in the simultaneous presence of 100 µM BL I and 2.5 µM phosphatase 1 and 2A inhibitor-OA. The experiment was conducted three times, and a representative example is shown

Effect of BL I and OA on Histone H1 Kinase and MAP Kinase Activities

We have previously demonstrated that treatment of cattle oocytes with OA accelerates both GVBD and histone H1 kinase activation and, moreover, that this treatment can partially overcome the inhibitory effect of cycloheximide and 6-dimethylaminopurine treatment on GVBD and activation of histone H1 kinase activity [42]. OA treatment can also induce MAP kinase activation in other mammalian species, such as the mouse or rat [25,43].

In contrast to the case in porcine oocytes (unpublished results), OA was not able to overcome the ability of BL I to inhibit either GVBD or histone H1 kinase and MAP kinase activities. Oocytes cultured in the presence of these two compounds for 24 h remained in the GV stage, although 72% displayed condensed chromosomes (Table 1). In addition, neither histone H1 kinase nor MAP kinase became activated after 8 h of BL I and OA treatment, whereas treatment with OA alone resulted in activation of these two enzymes by 8 h (Fig. 5).



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  		FIG. 5. Combined effect of BL I and OA on histone H1 and MAP kinase activities. A) Autoradiogram showing changes of histone H1 and MBP kinase activities in control oocytes (C0, C7, C20), oocytes cultured in the presence of 2.5 µM OA for indicated times (OA 2, 4, 6, 8), or oocytes cultured in the simultaneous presence of 2.5 µM OA and 100 µM BL I (OA/BL) for 2, 4, 6, and 8 h. B) Increased phosphorylation (and activation) of two MAP kinase family members (ERK1, 2) occurred in control oocytes cultured for 7 (C7) and 20 (C20) h, and in OA-treated oocytes after 6 (OA 6) and 8 (OA 8) h, but not in OA/BL I-treated oocytes, as revealed by mobility shifts on Western blot

Reversibility of BL I-Induced Inhibition of GVBD and Activation of Histone H1 Kinase and MAP Kinase

To ascertain whether the BL I-induced inhibition of GVBD was reversible, the oocytes were cultured in control or BL I-supplemented (100 µM) medium for 24 h. After this culture, one half of the oocytes (593) were fixed and stained for assessment of nuclear morphology, whereas the second half (600) were washed 3 times and transferred to medium containing BOS and gonadotropins and then cultured for an additional 24 h.

After the first 24 h of culture, 85–92% of oocytes in the control groups reached MII, except for oocytes cultured in follicular fluid, in which 68% of the oocytes remained at the late diakinesis-MI stage (Fig. 6A). As anticipated, inclusion of BL I in the medium inhibited meiotic maturation. The degree of BL I-induced inhibition depended on the medium; e.g., less inhibition was observed when BOS was included (Fig. 6A). It should be noted that while inclusion of FSH did not increase the incidence of progression to MII or overcome the BL I-induced inhibition of GVBD, inclusion of BL I in basic culture medium with PVA+FSH inhibited cumulus cell expansion, whereas cumulus cell expansion was only partly inhibited when follicular fluid was used as the medium for oocyte maturation (data not shown).



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  		FIG. 6. A) Morphology of bovine oocytes cultured for 24 h in the presence of BL I in different culture media. The oocytes were cultured either in control medium (basic medium supplemented with either 3 mg/ml BSA, or 3 mg/ml PVA and 100 ng FSH, or follicular fluid from < 5-mm follicles, or basic medium with 10% BOS) or in the same medium supplemented with 100 µM) BL I. A total of 593 oocytes (in 4 experiments) were cultured in control and BL I media for 24 h and were then fixed and stained for analysis of the morphology of nuclear maturation. a,b,c Different superscripts within columns GV or MII significantly differ. B) Reversibility of the BL I-mediated maturation block in different culture media (after 24-h culture with or without BL I followed by 24 h in control medium). A total of 600 bovine oocytes were cultured 24 h in control media and in media with BL I (see A), and subsequently 24 h in maturation medium with BOS and gonadotropins. a,b,c Different superscripts within columns MII significantly differ. LD, Late diakinesis

After transfer to, and culture in, BL I-free medium for another 24 h, it was apparent that the majority of oocytes resumed meiosis and progressed to MII (Fig. 6B). The highest degree of reversibility was observed when the oocytes were cultured in either basic culture medium with BSA or in follicular fluid (91.0 and 85.7%, respectively, matured to MII). The extent of oocytes that matured to MII in the other experimental groups was lower, especially in the group cultured with BL I and BOS, in which about 35% of oocytes became activated; i.e., they were either in telophase II, or a female pronucleus had formed.

To explore the possibility that the length of culture in BL I-containing medium could be extended to longer periods of time and still maintain a high degree of reversibility, oocytes were cultured for 48 h in BL I-containing medium before being transferred to BL I-free medium and cultured for an additional 24 h. When the oocytes were examined after the first 48 h of culture in BL I-containing medium, GVBD was markedly inhibited for oocytes cultured in basic culture medium containing either PVA or BSA (Fig. 7A). In contrast, oocytes cultured in basic culture medium containing BOS readily progressed to MII, and, in fact, ~25% became activated. When the GV-intact, BL I-inhibited oocytes were transferred to, and cultured in, BL I-free medium for an additional 24 h, the inhibitory effect was still reversible, and basic culture medium containing BSA, as before, supported the highest degree of reversibility (Fig. 7B). Oocytes cultured in basic culture medium containing BSA also displayed the best cumulus cell expansion, which was similar to that of oocytes cultured immediately after isolation (data not shown). It should be noted that in the control groups, i.e., oocytes cultured for a total of 76 h in medium that supports maturation, a high degree of activation was observed, except for oocytes cultured in follicular fluid.



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  		FIG. 7. A) Morphology of bovine oocytes cultured in the presence of BL I in different culture media for 48 h. Oocytes (519 from a total of 4 experiments) were cultured in control and BL I media for 48 h and then fixed and stained for the morphology of nuclear maturation. a,b,c Different superscripts within columns GV or MII significantly differ. B) Reversibility of the BL I-mediated maturation block in different culture media (after 24 h culture with or without BL I followed by 24 h in control medium). Oocytes (a total of 522) were cultured for 48 h in control media and media with BL I (A) and subsequently for 24 h in maturation medium with BOS and gonadotropins. a,b,c Different superscripts within columns MII significantly differ. *Degenerated GV oocytes. LD, Late diakinesis

The reversibility of the BL I-induced inhibition of GVBD was also linked to the activation of both histone H1 kinase and MAP kinase. As anticipated, oocytes cultured for either 24 or 48 h in medium containing BL I and remaining in the GV stage possessed low levels of these protein kinase activities (Fig. 8A, lanes 1, 3, 5, 11, 13, respectively), and immunoblot analysis with the anti-ERK1/ERK2 antibody documented that MAP kinase was completely dephosphorylated (Fig. 8B, lanes 1, 3, 5). In contrast, oocytes cultured in the basic culture medium with BSA only for 24 h (Fig. 8A, lane 2) or for 48 h (lane 10), or in the basic culture medium with PVA+FSH (lane 4) for 24 h, displayed high levels of both histone H1 and MBP kinase activities. Interestingly, control oocytes cultured in the basic culture medium with PVA+FSH for 48 h showed a decreased level of histone H1 kinase but a high level of MAP kinase activity (Fig. 8A, lane 12); the high level of MAP kinase activity was corroborated by immunoblot analysis with the anti-ERK1 and 2 antibodies, which revealed essentially total phosphorylation of ERK1 and 2 (Fig. 8A, lanes 2, 4).



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  		FIG. 8. Changes in histone H1 and MAP kinase activities in oocytes cultured in the presence of BL I and after its removal from control medium. A) Autoradiograms showing histone H1 kinase and MBP kinase activities in control (GV-stage) bovine oocytes (lane 1) and in bovine oocytes cultured in the presence (+) or absence (-) of BL I in media with BSA or with PVA/FSH for 24 h (lanes 2–5). Lanes 6–9, reversibility of the BL I block in above-mentioned groups after a subsequent 24-h culture in maturation medium with 10% BOS and gonadotropins. Lanes 10–13, histone H1 kinase and MBP kinase activities in bovine oocytes cultured in the presence or absence of BL I in different culture media for 48 h. Lanes 14–17, reversibility of the BL I block (of the above mentioned groups) analyzed after a subsequent 24-h culture in maturation medium containing 10% BOS and gonadotropins. B) Lanes 2–5, changes in phosphorylation status of ERK1 and ERK2 MAP kinases in bovine oocytes cultured in the presence (+) or absence (-) of BL I in media with BSA or with PVA/FSH for 48 h, observed as mobility shifts on Western blot. Lanes 6–9, reversibility of BL I effect on phosphorylation state of both MAP kinases after 24 h in maturation medium (same as in A)

Reversal of the BL I-induced inhibition after an initial 24 or 48 h of culture was accompanied by activation of both histone H1 kinase and MAP kinase activities (Fig. 8A, lanes 7, 9, 15, 17; Fig. 8B, lanes 7, 9), and the increase in activity was comparable to that observed in control oocytes (Fig. 8A, lanes 6, 8, 14; Fig. 8B, lanes 6, 8). It should be noted, however, that the level of activity of both histone H1 kinase and MAP kinase was lower in control oocytes that were first cultured for 48 h in the basic culture medium containing PVA+FSH and then cultured for an additional 24 h (Fig. 8A, lane 16 and 8B, lane 8).

Fertilization Competence of BL I-Inhibited and Matured Oocytes

The reversible nature of the BL I-induced inhibition of maturation raised the question whether the oocytes that matured to and arrested at MII were capable of being fertilized. Accordingly, the fertilization competence of oocytes cultured for 48 h in basic culture medium containing BSA and BL I and then cultured for an additional 20 h in BL I-free maturation medium that reached MII was assessed after fertilization in vitro (Table 2); control I oocytes were cultured for 48 h in basic culture medium with BSA without BL I and then an additional 20 h in maturation medium before fertilization; control II oocytes were permitted to resume meiosis immediately after their collection (20 h in maturation medium). Although both control groups and the experimental group displayed high levels of sperm penetration (82%, 90%, and 88%, respectively), there were striking differences in specific aspects of the fertilization process. For example, the incidence of polyspermy was much greater in the control I group compared to the BL I-treated group (53% versus 18%), and the incidence of normal pronucleus development was much lower in the control group (15% vs. 80%). Even when compared to control II, the experimental group showed a slightly higher incidence of normal pronucleus development (80% vs. 78%), and similarly the incidence of polyspermy was lower (18% versus 29). Thus, maintaining meiotic arrest in culture by addition of BL I before permitting oocyte maturation to occur by reversing this inhibition did not decrease the fertilization competence of such eggs, when compared to control oocytes.


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  		TABLE 2. Penetration, normal pronucleus development, and polyspermy in bovine oocytes


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we report that BL I can arrest meiotic maturation of bovine oocytes and that this arrest is correlated with inhibition of both MPF and MAP kinase activation, even though in assays in vitro using extracts prepared from MII eggs, BL I inhibits only MPF activity and not MAP kinase activity. Moreover, we demonstrate that this inhibition is reversible and can be exploited by using a two-step culture system that increases the developmental competence of the matured oocytes.

In our system, GVBD in cattle oocytes starts to occur by 7–8 h after initiation of culture, and by 12–14 h the oocytes reach MI. Subsequently, the synchronization of cell cycle progression decreases. Nevertheless, by 14–16 h most of the oocytes have reached anaphase I/telophase I, and by 24 h most of the oocytes have reached MII (data not shown). Correlated with GVBD and progression into MI is the activation of p34cdc2 kinase, which starts to increase by 7 h, and MAP kinase, which starts after about 8 h (Fig. 3). Others have reported that both kinases become activated after 9 h of culture and that this increase correlates with GVBD [26]. The activation of p34cdc2 kinase before that of MAP kinase is also observed in the mouse, in which the timing difference is more pronounced [35,44,45]. The temporal changes in p34cdc2 kinase that we report are also similar to those previously reported by others. For example, p34cdc2 kinase activity reached the first maximum after 12 h (15 h in [26]), at the time of MI; then it declined and reached a minimum level after 16 h (19 h in [26]) and rose again to second maximum after 24 h of culture. MAP kinase activity, on the other hand, reached maximum levels by 12 h and thereafter remained constant for the next 12 h, which is in good agreement with all data published for mammalian oocytes [26,27,35,44,45]. In contrast to a previous report [26], in which activation of a single protein of Mr = 42 000 identified as ERK2 was observed, we found that both ERK1 and ERK2 became activated, as evidenced by a decrease in electrophoretic mobility of both proteins.

We observed that activation of p34cdc2 kinase during cattle oocyte maturation occurred slightly before that of MAP kinase, and the activation of these kinases was tightly correlated with the initiation of GVBD. This temporal pattern of activation suggests that MAP kinase activation may be linked to p34cdc2 kinase activation. Consistent with this proposal is our observation that inhibition of p34cdc2 kinase by BL I treatment also resulted in the inhibition of MAP kinase activation. The inability of BL I to inhibit MAP kinase in extracts derived from MII-arrested eggs minimizes the likelihood that BL I can directly inhibit MAP kinase activation that occurs during maturation. In addition, the BL I-induced inhibition of GVBD of bovine oocytes was not overcome by treatment with OA; and in the oocytes treated simultaneously with BL I and OA, the activity of p34cdc2 kinase and MAP kinase remained low. This contrasts to the effect of OA treatment on porcine oocytes incubated in the presence of BL I, in which MAP kinase was activated in the absence of an increase in p34cdc2 kinase activity and in which GVBD occurred (unpublished results). In fact, MAP kinase is translocated to the GV and activated just before GVBD in porcine oocytes [46]. It should be noted that OA treatment can induce GVBD in both rat and mouse oocytes under conditions in which MAP kinase becomes fully activated while p34cdc2 kinase remains inactivated [25,43]. These results suggest that MAP kinase can substitute for the p34cdc2 kinase function in this process, even though MAP kinase activation in mouse oocytes normally occurs subsequently to GVBD [25,35] (as it also does during goat oocyte maturation [27].

Elevated levels of MAP kinase observed in MII eggs are strongly implicated in maintaining MII arrest and chromosomes in a condensed state; activation of MAP kinase is initiated by c-mos, which is an MAP kinase kinase kinase [47–49]. MAP kinase activity becomes elevated by MI and remains so during the MI to MII transition, whereas p34cdc2 kinase activity transiently decreases during this transition. Therefore, it has been suggested that MAP kinase (and not p34cdc2 kinase) is responsible for maintaining the microtubules in a metaphase state and the chromosomes in a condensed state [24,35]. MAP kinase is also associated with microtubule organizing centers at the spindle poles, suggesting a role in regulating spindle organization [24]. Our results, however, show that MAP kinase activity is not required for chromosome condensation, at least in the beginning of bovine oocyte maturation. When ovine oocytes were cultured in the presence of BL I, GVBD did not occur, and neither p34cdc2 kinase nor MAP kinase became activated even after 24 h of culture; and yet, fully condensed chromosomes were observed in these GV-intact oocytes. The process of chromosome condensation, however, most likely requires at least some changes in phosphorylation status, as the treatment of oocytes with 6-dimethylaminopurine can prevent chromosome condensation and can even cause decondensation of already condensed chromosomes [50]. The nature of the kinase(s) responsible for these processes is not known and will be the subject of our further studies. Guo et al. [51] have also shown in somatic cells that chromosome condensation during mitosis does not require either p34cdc2 kinase activity or histone H1 phosphorylation. Histone H2A and histone H3 instead become phosphorylated in these cells, however, only when phosphatases 1 and 2A have been inhibited by fostriecin or by OA.

The proposal that meiotic and developmental competence could be increased by prolonged culture in vitro of GV-intact oocytes is based on the positive effect of increasing the time during which oocytes are maintained in follicles after ovary isolation on their developmental competence following maturation in vitro [52]. In response to this observation, others have described two-step culture procedures in which spontaneous maturation is inhibited by either an inhibitor of protein synthesis (e.g., cycloheximide) or protein phosphorylation (e.g., 6-dimethylaminopurine) [53–56] before the inhibitor is removed and maturation is permitted to ensue. The specificity of BL I and the high degree of reversibility of oocyte maturation that is achieved after its removal from the culture system suggests that BL I treatment may be a much more effective method for increasing the meiotic and developmental competence of bovine oocytes after obtaining ovarian tissue. We are currently assessing our two-step culture system on the developmental competence of inseminated eggs derived from this procedure.


   ACKNOWLEDGMENTS
 
The authors are indebted to Mrs. J. Zelenková, Miss L. Málková, and Mr. S. Hladky for their skillful technical assistance. We also thank the companies Procházka and Vása from Roudnice n/L, Czech Republic, for donation of the bovine ovaries.


   FOOTNOTES
 
First decision: 16 August 1999.

1 This work was supported by the grant 524/96/K 162 of GA of the Czech Republic; FIRCA grant RO3-TW00691 (to M.K. and R.M.S.) and a grant from the NIH (HD 22681 to R.M.S.). Back

2 Correspondence: Richard Schultz, Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018. FAX: 215 898 8780; rschultz{at}mail.sas.upenn.edu Back

Accepted: September 15, 1999.

Received: June 30, 1999.


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