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Biology of Reproduction 65, 1751-1758 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Resumption of Meiosis Induced by Meiosis-Activating Sterol Has a Different Signal Transduction Pathway than Spontaneous Resumption of Meiosis in Denuded Mouse Oocytes Cultured In Vitro1

I. Faerge2,a,b, B. Terryc, J. Kalousd, P. Wahle, M. Lesslf, J.L. Ottesene, P. Hyttelb, and C. Grøndahla

a Fertility Team, SAC 2.02, Novo Nordisk A/S, 2820 Gentofte, Denmark b Department of Anatomy and Physiology, Royal Veterinary and Agricultural University, 1870 Frederiksberg C, Denmark c BioImage A/S, 2860 Søborg, Denmark d Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic e Pharmacology, Health Care Discovery, Novo Nordisk A/S, 2760 Måløv, Denmark f Research Laboratories, Schering AG, Berlin, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The sterol 4,4-dimethyl-5-cholesta-8,14,24-trien-3-ol (follicular fluid meiosis-activating sterol [FF-MAS]) isolated from human follicular fluid induces resumption of meiosis in mouse oocytes cultured in vitro. The purpose of this study was to examine the hypothesis that differential signal transduction mechanisms exist for FF-MAS-induced and spontaneous in vitro resumption of meiosis in mouse oocytes. Mouse oocytes were dissected from ovaries originating from mice primed with FSH 48 h before oocyte collection. Mechanically denuded germinal vesicle (GV) oocytes were in vitro matured in medium supplemented with hypoxanthine and FF-MAS or allowed to mature spontaneously; both groups were exposed to individual compounds known to inhibit specific targets in the cell. After 20–22 h of in vitro maturation, resumption of meiosis was assessed as the frequency of oocytes in GV breakdown (GVBD) stage. Pertussis toxin (2.5 µg/ml) did not influence resumption of meiosis in either group. Dibutyryl cyclic GMP (320 µM) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD, whereas the subtype 5 phosphodiesterase-inhibitor zaprinast (50 µM) inhibited GVBD in both groups. Microinjection of the catalytic subunit of cAMP-dependent protein kinase into oocytes inhibited spontaneous GVBD, but not FF-MAS-induced GVBD. An inhibitor of cytoplasmic polyadenylation, cordycepin (80 µM), inhibited or retarded spontaneous GVBD to a further extent than it did FF-MAS-induced GVBD. Spontaneous GVBD was more sensitive to the histone H1 kinase-inhibitor olomoucine (250 µM) than was FF-MAS-induced GVBD. Addition of the mitogen-activated protein kinase (MAPK)-inhibitor PD 98059 (50 µM), phospholipase C-inhibitor U-73122 (10 µM), p21ras-inhibitor lovastatine (250 µM), and the src-like kinase inhibitor PP2 (20 µg/ml) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD. Both MAPKs, extracellular regulated kinase (ERK) 1 and ERK2, were phosphorylated under FF-MAS-induced meiotic resumption, in contrast to spontaneous meiotic resumption, in which ERK1 and ERK2 phosphorylation occurred 2 h after GVBD. In the present study, we show that FF-MAS acts through an MAPK-dependent pathway, and we suggest that src-like kinase, p21ras, and phosphoinositide signaling lie upstream of MAPK in the FF-MAS-activated signaling pathway. Clearly, striking pathway differences are present between spontaneous versus FF-MAS-induced meiotic resumption.

meiosis, ovum, signal transducers, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fully grown mouse oocytes are arrested in the prophase of the first meiotic division within the ovarian follicle. In vivo, resumption of meiosis in oocytes occurs in response to the preovulatory surge of gonadotropins, especially LH, whereas in vitro, simple removal of oocytes from their inhibitory environment induces spontaneous resumption of meiosis in several species [1, 2]. The mechanism by which oocytes are inhibited from resuming meiosis in the ovarian follicular environment is not known. The follicular fluid contains purines (e.g., hypoxanthine) that are considered to be important inhibitory substances of oocyte maturation, at least in rodent oocytes [3–5]. Gap junctions connecting the oocyte with the surrounding cumulus cells within the follicle allow the transfer of small molecules from somatic cells to the germ cell. The inhibitory action has been attributed to the follicular wall [6], granulosa cells [7], and theca cells [8, 9], but the nature of the inhibitory mechanism has not yet been identified.

Similarly, the mechanism by which the oocyte overcomes meiotic arrest is not well understood. Several authors have reported evidence that cumulus cells can produce a gonadotropin-dependent, positive stimulus to meiotic resumption [10–12]. In 1995, certain sterols capable of inducing meiotic resumption were identified within the preovulatory follicle [13]. Follicular fluid meiosis-activating sterol (FF-MAS), identical to the sterol isolated from human follicular fluid, can induce, in a dose-dependent manner, in vitro resumption of meiosis in denuded and cumulus-enclosed mouse oocytes inhibited by hypoxanthine, 3-isobutyl-1-methylxanthine, or dibutyric cyclic AMP [14]. The origin of FF-MAS has not yet been established, but evidence suggests cumulus cells or follicle cells [11, 12]. In contrast to spontaneous in vitro meiotic resumption, FF-MAS-induced meiotic resumption in mouse oocytes is sensitive to cholera toxin [15], suggesting a possible role of a trimeric guanine nucleotide-binding protein (G protein)-coupled receptor in the FF-MAS signaling. Furthermore, FF-MAS signaling requires de novo protein synthesis, in contrast to spontaneous resumption of meiosis [15].

The second-messenger cAMP is involved in oocyte meiotic resumption. Several agents, such as cAMP derivatives [16] and phosphodiesterase (PDE) inhibitors [17], can modulate the intraoocyte cAMP level and, subsequently, meiotic resumption. The purine hypoxanthine is present at concentrations of 2–4 mM in mouse follicular fluid [4], where it maintains oocytes in meiotic arrest by inhibition of cAMP-dependent PDE activity [17]. The PDEs constitute a large group of enzymes that belong to at least 10 gene families. Subtype 3 PDE (PDE3) and subtype 4 PDE (PDE4) are thought to be selectively expressed and regulated in the cumulus-oocyte complex (COC). Subtype 3 PDE is predominant in the oocyte, whereas PDE4 is expressed mainly in the granulosa cells. It has been suggested that meiotic resumption requires high cAMP levels in the granulosa cells and low or decreasing levels in the oocyte, and that such opposing levels of cAMP may result from the selective expression and regulation of these PDEs in the two compartments of the COC [18].

The resumption of meiotic maturation in oocytes involves changes in the phosphorylation state of a series of specific proteins with kinase and phosphatase activity [19–21]. Among these is the cytoplasmic factor, maturation-promoting factor (MPF), which leads to germinal vesicle (GV) breakdown (GVBD). The MPF is a heterodimer of a regulatory subunit, cyclin B, and a catalytic subunit, p34cdc2. Prophase-arrested mouse oocytes contain complexes of cyclin B and p34cdc2 that can be activated by a dephosphorylation process, thereby probably triggering histone H1 kinase activity [22]. Also, mos and enzymes of the mitogen-activated protein kinase (MAPK) family are involved in meiotic maturation of oocytes. The MAPK, also called extracellular regulated kinase (ERK), is a serine/threonine kinase activated by the c-mos protooncogene protein kinase during mouse oocyte maturation [23, 24].

The aim of this study was to examine the hypothesis that a differential signal transduction pathway exists for FF-MAS-induced versus spontaneous resumption of meiosis. The FF-MAS-induced and spontaneous in vitro meiotic resumption was monitored in denuded mouse oocytes as GVBD after exposure to a series of specific inhibitor compounds, and MAPK phosphorylation was assessed in FF-MAS-induced and spontaneous GVBD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis, Purification, and Identification of FF-MAS

The FF-MAS was synthesized by a medicinal chemistry route by the Department of Medicinal Chemistry, Novo Nordisk A/S, Denmark [25].

Oocyte Collection and Culture

Oocytes were obtained from immature female mice (C57BL/6J x DBA/2J F1; M&B, Ry, Denmark) weighing 11–17 g and kept under controlled temperature, light, and relative humidity. The mice were subjected to an i.p. injection of 0.2 ml of gonadotropins (Gonal F; Serono, Randolph, ME) containing 20 IU of FSH and were killed by cervical dislocation 48 h later. After removal of ovaries, oocytes were recovered in Petri dishes under a stereomicroscope into {alpha}-minimum essential medium (without ribonucleosides, cat. no. 22561; Gibco BRL, Grand Island, NY) supplemented with 3 mg/ml of human serum albumin (HSA; State Serum Institute, Copenhagen, Denmark), 0.23 mM sodium pyruvate (cat. no. S-8636, Sigma-Aldrich Ltd., Irvine, UK), 2 mM glutamine (cat. no. 16-801-49; ICN Biochemicals, Inc., Aurora, OH), 100 IU/ml of penicillin, and 100 µg/ml of streptomycin (cat. no. 16-700-49; ICN Biochemicals) with 3 mM hypoxanthine (cat. no. H-9377; Sigma-Aldrich Chemie Gmbh, Steinheim, Germany). This medium was designated as Hx medium. The above-described medium without hypoxanthine was designated as Hx-free medium. The FF-MAS (NNC 54-0092, batch 18H; Novo Nordisk, Måløv, Denmark) was applied at a concentration of 5 or 10 µM in Hx medium and designated as Hx+FF-MAS. For in vitro maturation, Hx medium, Hx+FF-MAS, and Hx-free medium were supplemented with 5 mg/ml of HSA. After mechanical removal of cumulus cells, approximately 300 oocytes from 14 to 16 mice each day were pooled together and washed three times. Denuded spherical oocytes of uniform size containing an intact GV were allocated to treatment groups and cultured in 400 µl of medium in four-well dishes (Nunclon, Roskilde, Denmark) at 37°C in 5% CO2 in humidified air.

For immunoblotting, oocytes were collected in aliquots of 10 oocytes in 2–5 µl of PBS. Control GV-stage oocytes were collected before onset of in vitro maturation. Denuded oocytes were allowed to mature spontaneously. After 90 min of culture in Hx-free medium, all GV-stage oocytes were isolated, and those oocytes undergoing GVBD within the next 30 min were either collected for analysis or cultured for 2 and 4 h post-GVBD stage and to the metaphase II stage, then collected for analysis. The FF-MAS-induced oocytes were collected in a different manner due to the protarded maturation kinetics of FF-MAS-induced meiotic resumption compared to those of spontaneous meiotic resumption [26]. Seven hours after onset of culture, GV oocytes were isolated, and from those oocytes going into GVBD within the next 120 min, samples of 10 oocytes were collected. The remaining GVBD oocytes were allowed to progress further in meiosis, and samples were collected 2 and 4 h after GVBD and at metaphase II. To evaluate possible differences in MAPK phosphorylation over time, samples of FF-MAS-maturing oocytes were collected as described above, but starting at 9 and 11 h after onset of culture. Furthermore, the following control oocytes were collected: GV-stage oocytes in Hx medium and GV-stage oocytes in Hx+FF-MAS at 8, 10, and 12 h.

GVBD Assay

The in vitro culture assay for resumption of meiosis was conducted as previously reported [14]. Briefly, progression of maturation after each treatment was assessed by the presence of GV or its absence (i.e., GVBD). The percentage of oocytes in a well that had undergone GVBD (%GVBD) was defined as the number of oocytes in GVBD stage divided by the total number of oocytes multiplied by 100 in that well. For in vitro maturation, Hx medium, Hx+FF-MAS, and Hx-free medium were supplemented with the respective inhibitor compounds, and approximately 40 oocytes were cultured in each replicate. The concentration of each inhibitor compound found to exert a differential inhibitory effect on spontaneous and FF-MAS-induced resumption of meiosis was found in preliminary experiments.

Stock solutions of 8 mM cordycepin (cat. no. C-3394; Sigma-Aldrich Chemie Gmbh), 16 mM lovastatine (cat. no. 438185; Calbiochem-Novabiochem Corporation, La Jolla, CA), and 3 mM U-73122 (cat. no. 662035-S; Calbiochem-Novabiochem) were prepared in ethanol and diluted in Hx medium, Hx+FF-MAS, and Hx-free medium to final concentrations of 80 and 200 µM. Stock solutions of 20 mM zaprinast (cat. no. Z-0878; Sigma-Aldrich Chemie Gmbh), 25 mM olomoucine (cat. no. 495620-S; Calbiochem-Novabiochem), 50 mM PD 98059 (cat. no. 513000-S; Calbiochem-Novabiochem), 13 mM PP2 (cat. no. 529573; Calbiochem-Novabiochem), and 3 mM U-73343 (cat. no. 662041; Calbiochem-Novabiochem) were prepared in dimethyl sulfoxide (DMSO) and diluted in Hx medium, Hx+FF-MAS, and Hx-free medium to final concentrations of 50 µM zaprinast, 250 µM olomoucine, 50 µM PD 98059, and 65 µM PP2, respectively. Stock solutions of 16 mM dibutyryl cyclic GMP (dbcGMP) (cat. no. D-3510; Sigma-Aldrich Chemie Gmbh) and 1.0 mg/ml of pertussis toxin (cat. no. 516560; Calbiochem-Novabiochem) were prepared in distilled water and diluted to 320 µM and 2.5 µg/ml, respectively, in Hx-free medium and Hx+FF-MAS. Preincubation of oocytes involved 2 h (zaprinast, PD-98059, and PP2), 3 h (pertussis toxin), or 4 h (lovastatine and dbcGMP) of incubation in Hx medium containing the respective inhibitor before transfer into Hx-free medium or Hx+FF-MAS plus inhibitor. For each compound tested, control groups of approximately 40 oocytes were cultured in Hx-free medium, Hx medium, and Hx+FF-MAS under culture conditions identical to those in the treatment groups. Addition of ethanol, distilled water, or DMSO at the concentrations used in each experiment did not affect control groups.

Microinjection of the Catalytic Subunit of Protein Kinase A in Denuded Mouse Oocytes

The GV oocytes were microinjected with the catalytic subunit of cAMP-dependent protein kinase (PKA; Promega, Madison, WI) using a piezo-driven injection pipette. The catalytic subunit of PKA was diluted in a KH2PO4 plus dithiothretol buffer. Each oocyte was injected with approximately 10 pl of a 0.22–0.66 U/µl concentration of the catalytic subunit of PKA. Resumption of meiosis was triggered by transfer of the injected oocytes from Hx medium to Hx-free medium or by addition of 10 µM FF-MAS to the Hx medium.

SDS-PAGE and Immunoblotting

The previously described samples of oocytes were washed five times in PBS and stored immediately at -80°C until electrophoresis. The samples were lysed in 10 ml of double-strength SDS sample buffer containing 5% 2-mercaptoethanol and separated on 9% SDS-PAGE gels 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-Aldrich Chemie Gmbh) dissolved in 0.05% Tween-20 in Tris-buffered saline (pH 7.4; TTBS) for 1 h before development with anti-ERK1 antibody (sc-94, 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by secondary anti-rabbit, horse radish peroxidase-linked immunoglobulin (1:5000; Amersham Pharmacia Biotech, Freiburg, Germany); the blots were incubated with each antibody for 1 h at room temperature. The blots were then washed at least five times for at least 10 min each wash in TTBS, then developed with an enhanced chemiluminescence kit (Amersham) according to the manufacturer's instructions.

Statistical Analysis

In the GVBD assay and the microinjection experiment, each experiment was performed at least three times. Data from three replicates were pooled and analyzed by chi-square test. Results were considered to be statistically significant at P < 0.05. Percentage data in the text are shown as the mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pharmacological Inhibitors on FF-MAS-Induced and Spontaneous GVBD

It has previously been demonstrated that FF-MAS-induced GVBD, but not spontaneous GVBD, is sensitive to cholera toxin [15], suggesting a role of G proteins in the signal transduction mechanism of FF-MAS. To explore further the role of G proteins, pertussis toxin was applied to maturing oocytes that were allowed to mature spontaneously or induced by FF-MAS. When pertussis toxin was applied, no effect was observed in %GVBD in Hx+FF-MAS-cultured oocytes compared to the Hx+FF-MAS control group (86.3% ± 1.6% vs. 86.2% ± 1.7%) or in spontaneously maturing oocytes compared to the Hx-free control group (91.0% ± 0.8% vs. 90.97percnt; ± 1.2%) (Table 1).


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  		TABLE 1. Influence of selected inhibitory compounds on FF-MAS-induced and spontaneous meiotic resumption, respectively, in denuded mouse oocytes after 20–22 h of in vitro culture

Because cGMP can delay spontaneous maturation in rat oocytes [27] and the cAMP:cGMP ratio increases before oocyte maturation [28], we explored possible roles of cGMP by using dbcGMP and the fairly specific inhibitor of PDE5, zaprinast, which is a PDE5 antagonist in somatic cells [29]. When dbcGMP was added to Hx+FF-MAS-cultured oocytes, %GVBD was significantly lower than in the Hx+FF-MAS control group (21.9% ± 8.0% vs. 82.4% ± 5.1%) (Table 1). When oocytes were preincubated with dbcGMP for 4 h in Hx medium and then transferred to Hx-free medium containing dbcGMP, the percentage of spontaneous GVBD was not lower than in the Hx-free control group (84.8% ± 2.6% vs. 91.3% ± 3.3%) (Table 1). When zaprinast was added to Hx+FF-MAS-treated oocytes, the %GVBD was significantly lower than in the Hx+FF-MAS control (19.3% ± 4.8% vs. 86.9% ± 3.4%) (Table 1). When spontaneously maturing oocytes were preincubated with zaprinast for 2 h in Hx medium and then transferred to Hx-free medium containing zaprinast, a significantly lower %GVBD was observed than in the Hx-free control group (19.7% ± 2.0% vs. 90.5% ± 2.9%) (Table 1).

In oocytes, maternal mRNA translation is regulated largely by cytoplasmic polyadenylation. Activation of MPF requires cytoplasmic polyadenylation [30], a process that can be inhibited by cordycepin [31–33]. Therefore, the sensitivity of FF-MAS-induced and spontaneous GVBD to the inhibitor of cytoplasmic polyadenylation, cordycepin, was assessed, and a modest decrease in %GVBD was found when cordycepin was added to Hx+FF-MAS-cultured oocytes compared to the Hx+FF-MAS control group (72.5% ± 3.8% vs. 88.4% ± 2.6%), whereas in spontaneously maturing oocytes, %GVBD was significantly lowered by cordycepin (23.1% ± 7.2% vs. 93.8% ± 0.8%) (Table 1). Thus, the sensitivity to cordycepin was significantly different between spontaneous and FF-MAS-induced maturation.

We also wanted to explore the role of MPF and MAPK in oocyte maturation. Olomoucine inhibits or retards the prophase-to-metaphase I transition in oocytes of several species, including the mouse [34], by targeting histone H1 kinase [35, 36]. Therefore, olomoucine was used to elucidate possible differences in the involvement of histone H1 kinase during spontaneous and FF-MAS-induced GVBD. When olomoucine was added to Hx+FF-MAS-cultured oocytes, %GVBD was only slightly lower than in the Hx+FF-MAS control group (73.6% ± 6.9% vs. 88.1% ± 2.9%). Spontaneous %GVBD, however, was significantly different from that in the Hx-free control group when olomoucine was added to the culture (51.3% ± 10.4% vs. 88.4% ± 1.8%) (Table 1). When oocytes were preincubated in Hx medium with an inhibitor of MAPK activation, PD 98059, for 2 h and then transferred to Hx+FF-MAS and PD 98059 for 20 h of in vitro culture, the %GVBD was significantly lower than in the Hx+FF-MAS control group (20.9% ± 2.1% vs. 81.1% ± 7.3%), whereas in oocytes transferred to Hx-free medium and PD 98059, the %GVBD was not significantly different from that in the Hx-free control (81.4% ± 2.9% vs. 86.5% ± 3.9%) (Table 2). Thus, the sensitivity to the MAPK inhibitor PD 98059 was high in the FF-MAS-induced maturation, whereas no effect was seen in spontaneous maturation.


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  		TABLE 2. Influence of inhibitors of MAPK and src-like kinase on FF-MAS-induced and spontaneous meiotic resumption in denuded oocytes after 20–22 h of in vitro culture

Because inhibition of phosphoinositol metabolism blocks FSH-induced resumption of meiosis [37], we tested the ability of the inhibitor of phospholipase C, U-73122, to inhibit GVBD. We found that FF-MAS-induced GVBD was sensitive to this inhibitor (14.6% ± 10.4% vs. 76.0% ± 3.6%), whereas spontaneous GVBD was not (78.7% ± 0.6% vs. 83.7% ± 3.2%) (Table 1). In contrast, the weak analogue U 73343 did not inhibit either FF-MAS-induced or spontaneous GVBD at concentrations that had toxic effects on the oocytes.

The protein p21ras is an upstream activator of MAPK in Xenopus oocytes [38, 39], in which it can induce meiotic maturation [40], so we tested the influence of the inhibitor of p21ras, lovastatine, on FF-MAS-induced and spontaneous GVBD. When denuded oocytes were preincubated with lovastatine for 4 h in Hx medium and then exposed to Hx+FF-MAS and lovastatine for 20 h, %GVBD was significantly lower than in the Hx+FF-MAS control group (18.7% ± 6.1% vs. 82.3% ± 3.1%) (Table 1). When oocytes were transferred to Hx-free medium and lovastatine and then cultured in vitro for 20 h after 4 h of preincubation in Hx medium with lovastatine, no difference in %GVBD was observed compared to the Hx-free control group (84.3% ± 4.3% vs. 92.7% ± 1.9%) (Table 1). Again, these results suggest involvement of an MAPK-activated pathway in FF-MAS-induced maturation, in contrast to spontaneous maturation.

In somatic cells, src-like kinase has been demonstrated recently to act downstream from the G protein ß{gamma} subunit and to play an important role in relaying signals from the G protein-coupled receptor (GPCR) to MAPK [41, 42]. We used the inhibitor of src-like kinase, PP2, to test the possibility that src-like kinase has a role in signal transduction in oocytes. When oocytes were preincubated for 2 h with PP2 in Hx medium and then exposed to Hx+FF-MAS and PP2 for 20 h of in vitro culture, %GVBD was significantly lower than in the Hx+FF-MAS control group (26.9% ± 2.9% vs. 80.6% ± 4.1%) (Table 2). When oocytes were transferred to Hx-free medium and PP2 after 2 h of preincubation in Hx medium with PP2, no significant difference in %GVBD was observed compared to the Hx-free control (70.94% ± 8.6% vs. 81.9% ± 2.8%) (Table 2).

Microinjection of the Catalytic Subunit of PKA on GVBD in Spontaneous and FF-MAS-induced GVBD

Microinjection of oocytes with 0.22 U/µl of the catalytic subunit of PKA inhibited resumption of spontaneous GVBD in 18 of 22 oocytes (18% GVBD), compared to 2 of 62 oocytes (97% GVBD) in the spontaneous control group. In contrast, no inhibition was observed in FF-MAS-induced maturation when 0.22, 0.44, or 0.66 U/µl were injected (100%, 82%, and 83% GVBD, respectively) (Table 3). Thus, the catalytic subunit of PKA could significantly inhibit spontaneous oocyte maturation, whereas FF-MAS-induced maturation was unaffected.


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  		TABLE 3. Differential effect of microinjected catalytic subunit of PKA on inhibitory action on spontaneous versus FF-MAS-induced oocyte maturation.a

MAPK Phosphorylation in Spontaneous and FF-MAS-Induced GVBD

In spontaneously maturing oocytes, phosphorylation of p42 and p44 MAPK occurred 2 h after GVBD (Fig. 1a, lane 6) and remained phosphorylated 2 and 4 h after GVBD (Fig. 1b, lanes 3 and 4) and at metaphase II (Fig. 1a, lane 10), as observed by a mobility shift of p42 and p44 MAPK. In FF-MAS-induced GVBD, phosphorylation of p42 and p44 MAPK occurred concomitant with GVBD (Fig. 1a, lanes 7–9). In oocytes cultured for 2 and 4 h after the GVBD stage and in metaphase II oocytes, p42 and p44 MAPK were fully phosphorylated in both spontaneous (Fig. 1b, lanes 3, 4, and 11) and FF-MAS-induced meiotic resumption (Fig. 1b, lanes 5–10). Thus, MAPK phosphorylation occurred during FF-MAS-induced resumption of meiosis, in contrast to spontaneous maturation, in which MAPK phosphorylation occurred as a clear, post-GVBD event.



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  		FIG. 1. MAPK phosphorylation in FF-MAS-induced and spontaneous GVBD in denuded mouse oocytes. In FF-MAS-induced GVBD, phosphorylation of p42 and p44 MAPK occurred during GVBD (a, lanes 7–9) as observed by a mobility shift of p42/p44 and remained phosphorylated 2 h (b, lanes 5, 7, and 9) and 4 h after GVBD (b, lanes 6, 8, and 10) and at M-II (b, lane 12). In spontaneously maturing oocytes, no phosphorylation of p42 and p44 MAPK occurred at GVBD (b, lane 2), whereas phosphorylation of p42 and p44 MAPK occurred 2 h after GVBD (b, lane 3) and remained phosphorylated 4 h after GVBD (b, lane 4) and at metaphase II (b, lane 11). K0 GV, 0 h GV-stage oocytes; K4sp GV, GV-stage oocytes after 4 h culture in Hx-free medium; 8h, 10h, and 12h MAS GV, GV-stage oocytes cultured for 8, 10, and 12 h, respectively, in Hx+FF-MAS; Spon GVBD, GVBD-stage oocytes cultured in Hx-free medium; 8h, 10h, and 12h MAS GVBD, oocytes in GVBD stage at 8, 10, and 12 h, respectively, after culture in Hx+FF-MAS; Spon M-II, oocytes cultured to metaphase II in Hx-free medium; MAS M-II, oocytes cultured to metaphase II in Hx+FF-MAS; Spon GVBD +2h and +4h, GVBD-stage oocytes cultured for an additional 2 and 4 h, respectively, after GVBD in Hx-free medium; MAS 8h, 10h, and 12h GVBD +2h and +4h, GVBD-stage oocytes at 8, 10, and 12 h cultured for an additional 2 and 4 h, respectively, in Hx+FF-MAS


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, clear and distinct differences were observed in the signaling pathway of FF-MAS-induced meiotic resumption compared to spontaneous resumption of meiosis in denuded mouse oocytes. We wanted to observe and detail whether FF-MAS-induced maturation and spontaneous GVBD make use of the same signaling pathway or whether discrepancies could be found.

We previously demonstrated that FF-MAS-induced, but not spontaneous, GVBD is sensitive to cholera toxin [15], suggesting a role of G proteins in the signal transduction mechanism of FF-MAS. Because the intraoocyte cAMP level decreases during spontaneous GVBD, FF-MAS could possibly induce GVBD by a similar mechanism through a pertussis toxin-sensitive GPCR. Pertussis toxin catalyzes the ADP ribolysation of inhibitory G protein (Gi protein), thus uncoupling the receptor from the Gi protein. Hence, pertussis toxin would be expected to block FF-MAS-induced meiotic resumption if FF-MAS mediates its effect through adenylate cyclase. Our results suggest that, if FF-MAS acts through a receptor linked to a G protein, it is probably not a pertussis toxin-sensitive G protein.

It has been demonstrated that cGMP can delay spontaneous maturation in rat oocytes [27], and that the cAMP:cGMP ratio increases before oocyte maturation [28]. We explored possible roles of cGMP by using dbcGMP and the fairly specific inhibitor of PDE5, zaprinast, which is a PDE5 antagonist in somatic cells [29]. In somatic cells, the biological effect of cGMP may be mediated through the competitive inhibition of PDE3 [43], leading to an increased cAMP level [44]. That GVBD induced by FF-MAS was inhibited by dbcGMP, whereas spontaneous GVBD was not, may reflect that FF-MAS-maturing oocytes, because they are cultured in the presence of hypoxanthine, are more sensitive to an elevation in the intraoocyte cAMP level than spontaneously maturing oocytes cultured without hypoxanthine. Previously, an inhibitory effect on oocyte maturation of zaprinast has been found only at a very high concentration (median inhibitory concentration [IC50], 200 µM), with the effect being attributed to an unspecific action of zaprinast [45]. In our studies, both spontaneous and FF-MAS-induced GVBD were equally sensitive to zaprinast at the lower concentration of 50 µM. This leads us to conclude that cGMP may inhibit a step leading to GVBD that is common to both spontaneous and FF-MAS-induced maturation. Zaprinast may be more efficient than dbcGMP in producing the required local elevation of cGMP needed to inhibit GVBD. Many questions concerning the role of cGMP in resumption of meiosis remain to be answered, and further studies are required to establish whether a zaprinast-sensitive cGMP-PDE is actually expressed in the oocyte and, if so, to establish its possible role in GVBD.

The data presented here suggest that both FF-MAS-induced and spontaneous GVBD are sensitive to changes in intraoocyte levels of cAMP, but not to the same extent, possibly because of the presence of hypoxanthine in the culture medium of FF-MAS-matured oocytes. To explore the participation of PKA in FF-MAS-induced GVBD, hypoxanthine-arrested oocytes were microinjected with the catalytic subunit of PKA and allowed to undergo spontaneous or FF-MAS-induced GVBD. We found, in accordance with previous results, that spontaneously maturing oocytes were inhibited by injection of the catalytic subunit of PKA [46], whereas FF-MAS-induced GVBD was not blocked by the catalytic subunit of PKA. These data suggest that the signaling pathway of FF-MAS-induced GVBD diverges from that of spontaneous GVBD downstream of cAMP but upstream of PKA. If cAMP is not involved in the FF-MAS signaling, then our results may reflect a modulation of a cAMP/PKA-dependent inhibitory principle in the resumption of meiosis. Then, FF-MAS may act through a signaling cascade independent of the inhibitory cascade, with the two cascades converging at a later point (e.g., at the level of MPF activation).

Maternal mRNA translation is regulated largely by cytoplasmic polyadenylation. Activation of MPF requires cytoplasmic polyadenylation [30], a process that can be inhibited by cordycepin [31–33]. We found that spontaneous GVBD was sensitive to cordycepin, which is in accordance with the findings of Osborn and Moor [47] in ovine oocytes. In contrast, FF-MAS-induced GVBD was only slightly modulated by cordycepin. These data further support the hypothesis that FF-MAS-mediated and spontaneous GVBD are regulated differently.

Olomoucine was used explore the role of MPF activation in FF-MAS-induced GVBD, because it targets histone H1 kinase [35, 36]. Olomoucine inhibits or retards the prophase-to-metaphase I transition in oocytes of several species, including the mouse [34]. Accordingly, we found that olomoucine, albeit at a very high concentration, reduced the rate of spontaneous GVBD by 36%, whereas the rate of FF-MAS-induced GVBD was reduced by 14%. Olomoucine is a selective inhibitor of cdc and cdk protein kinases in the range of 3–7 µM, with an IC50 of 7 µM for p34cdc2/cyclin B and of 25 µM for p44 MAPK and no significant effect on other protein kinases at 1 mM [34–36, 48] (Table 4). Despite the high concentration of olomoucine, the effect is unlikely to be due to p42 MAPK inhibition, because the PD 98059 trial does not have the same profile as the olomoucine trial.


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  		TABLE 4. The IC50 values of some inhibitor compounds used in this study

Activation of MAPK during meiotic resumption has been established in oocytes from several species, including the mouse, pig, and cow [23, 49, 50]. That FF-MAS was less sensitive than spontaneously maturing oocytes to olomoucine led us to test the role of MAPK in FF-MAS-induced GVBD, because MAPK and MPF have previously been demonstrated to display substrate overlap [51]. Our observations in the PD 98059 trial showed that activation of MAPK is a prerequisite for FF-MAS-induced resumption of meiosis, whereas spontaneous GVBD can occur in an MAPK-independent way. Mos knock-out female mice have very reduced fertility [52, 53]. In vitro, oocytes from c-mos-deficient mice can complete first meiotic division but fail to arrest at metaphase II, resulting in a substantial decrease in the fertilization rate [52, 53]. Additionally, abnormal spindles and loosely condensed chromosomes have been observed in mos-deficient oocytes [54]. These results suggest that mos/MAPK mediate crucial events following GVBD; however, these events are not a prerequisite to spontaneous, in contrast to FF-MAS-induced, GVBD. The earlier activation of MAPK in FF-MAS-matured oocytes compared to spontaneous GVBD may lead to a more developmentally competent oocyte and, thus, account for the higher in vitro fertilization rate observed in FF-MAS-treated compared to spontaneously matured oocytes [55]. This apparent paradox—FF-MAS signaling is mediated through MAPK activation, whereas mos-deficient oocytes can resume meiosis in vivo without MAPK activation—may be explained by the existence of different signal transduction pathways in the oocyte leading to GVBD. In 3-isobutyl-1-methylxanthine-arrested mouse oocytes, overexpression of c-mos or MAPK in the absence of MPF leads to a prolonged meiosis I, with localized nuclear envelope disassembly, partial chromosome condensation, and formation of microtubule arrays [56]. This study suggests that, although activation of MAPK appears to be essential in FF-MAS-mediated resumption of meiosis, MPF must be activated at the same time or subsequent to MAPK activation to secure normal progression of meiosis. Because the role of mos/MAPK has been attributed to regulation of spindle assembly and/or function [23, 57, 58], it is interesting that the number of small, cytoplasmic, aster-like microtubular aggregates was significantly elevated in FF-MAS-matured metaphase I mouse oocytes compared to spontaneously matured oocytes [26].

In somatic cells, activation of certain GPCR can stimulate MAPK through a ras- and raf-dependent pathway [59, 60] in which phosphoinositide 3 kinase is also involved [61]. Recently, it was demonstrated that inhibition of the phosphoinositide metabolism inhibited FSH-induced, but not spontaneous, meiotic resumption in mouse oocytes [37]. In accordance with these results, we found that inhibition of phospholipase C blocked FF-MAS-induced, but not spontaneous, meiotic resumption. Because U-73122, an inhibitor of phospholipase C, can inhibit signaling through known G protein-coupled receptors, its action may indicate such a signaling pathway involved in FF-MAS-induced resumption of meiosis. Previously, p21ras has been shown to act as an upstream activator of MAPK in Xenopus laevis oocytes [38, 39] and to induce meiotic maturation in this species [40, 62]. In accordance with these findings, we found that FF-MAS-induced GVBD is sensitive to inhibition of p21ras and to inhibition of phosphoinositide hydrolysis. In somatic cells, src-like kinase has been demonstrated recently to act downstream from the G protein ß{gamma} subunit and to play an important role in relaying signals from GPCR to MAPK [41, 42]. An inhibitor of src-like kinases, PP2, was able to inhibit FF-MAS-induced, but not spontaneous, GVBD, indicating that src-like tyrosine kinase is involved in the postreceptor signaling mechanism of FF-MAS, but not in spontaneous GVBD.

We show in the present study that FF-MAS acts through an MAPK-dependent pathway, and we suggest that src-like kinase, p21ras, and phosphoinositide signaling lie upstream of MAPK in the FF-MAS-activated signaling pathway. All the steps appear to be necessary, because inhibition of one can completely block the pathway. However, a conclusion as to whether the steps are activated sequentially or in parallel pathways cannot be drawn from the data presented. Lack of target specificity of some of the inhibitors used makes it difficult to identify the precise signaling processes involved in the action of FF-MAS on meiotic resumption. However, striking pathway differences clearly are present between spontaneous versus FF-MAS-induced resumption of oocyte meiosis.


   ACKNOWLEDGMENTS
 
The authors wish to thank Mrs. Elene J. Carlsen and Mrs. Tina Olesen for their skillful technical assistance.


   FOOTNOTES
 
First decision: 17 December 1999.

1 Supported by grant 524/98/0231 from Grant Agency of the Czech Republic to J.K. Back

2 Correspondence: Inger Faerge, Fertility Team, SAC 2.02, Novo Nordisk A/S, Sauntesvej 13, 2820 Gentofte, Denmark. FAX: 45 44431063; ingf{at}novonordisk.com Back

Accepted: July 18, 2001.

Received: November 9, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pincus G, Enzmann EV. The comparative behavior of mammalian eggs in vivo and in vitro. I. The activation of ovarian eggs. J Exp Med 1935; 62:655-675
  2. Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 1965; 208:349-351[CrossRef][Medline]
  3. Downs SM, Coleman DL, Ward-Bailey PF, Eppig JJ. Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight fraction of porcine follicular fluid. Proc Nat Acad Sci U S A 1985; 82:454-458[Abstract/Free Full Text]
  4. Eppig JJ, Ward-Bailey PF, Coleman DL. Hypoxanthine and adenosine in murine ovarian follicular fluid: concentrations and activity in maintaining oocyte meiotic arrest. Biol Reprod 1985; 33:1041-1049[Abstract]
  5. Downs SM. Purine control of mouse oocyte maturation—evidence that nonmetabolized hypoxanthine maintains meiotic arrest. Mol Reprod Dev 1993; 35:82-94[CrossRef][Medline]
  6. Leibfried L, First NL. Effect of bovine and porcine follicular fluid and granulosa cells on maturation of oocytes in vitro. Biol Reprod 1980; 23:699-704[Abstract]
  7. Tsafriri A, Channing CP. An inhibitory influence of granulosa cells and follicular fluid upon porcine oocyte meiosis in vitro. Endocrinology 1975; 96:922-927[Abstract]
  8. Richard FJ, Sirard MA. Effects of follicular cells on oocyte maturation. 1. Effects of follicular hemisections on bovine oocyte maturation in vitro. Biol Reprod 1996; 54:16-21[Abstract]
  9. Richard FJ, Sirard MA. Effects of follicular cells on oocyte maturation. 2. Theca cell-inhibition of bovine oocyte maturation in vitro. Biol Reprod 1996; 54:22-28[Abstract]
  10. Downs SM, Daniel SAJ, Eppig JJ. Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic cell origin. J Exp Zool 1988; 245:86-96[CrossRef][Medline]
  11. Guoliang X, Byskov AG, Andersen CY. Cumulus cells secrete a meiosis-inducing substance by stimulation with forskolin and dibutyric cyclic adenosine-monophosphate. Mol Reprod Dev 1994; 39:17-24[CrossRef][Medline]
  12. Byskov AG, Andersen CY, Hossaini A, Guoliang X. Cumulus cells of oocyte-cumulus complexes secrete a meiosis-activating substance when stimulated with FSH. Mol Reprod Dev 1997; 46:296-305[CrossRef][Medline]
  13. Byskov AG, Andersen CY, Nordholm L, Thøgersen H, Guoliang X, Wassmann O, Andersen JV, Guddal E, Roed T. Chemical-structure of sterols that activate oocyte meiosis. Nature 1995; 374:559-562[CrossRef][Medline]
  14. Grøndahl C, Ottesen JL, Lessl M, Faarup P, Murray A, Grønvald FC, Hegele-Hartung C, Ahnfelt-Rønne I. Meiosis-activating sterol promotes resumption of meiosis in mouse oocytes cultured in vitro in contrast to related oxysterols. Biol Reprod 1998; 58:1297-1302[Abstract/Free Full Text]
  15. Grøndahl C, Lessl M, Faerge I, Hegele-Hartung C, Wassermann K, Ottesen JL. Meiosis activating sterol mediated resumption of meiosis in mouse oocytes in vitro is influenced by protein synthesis inhibition and cholera toxin. Biol Reprod 2000; 62:775-780[Abstract/Free Full Text]
  16. Cho WK, Stern S, Biggers JD. Inhibitory effect of dibutyric cAMP on mouse oocyte maturation in vitro. J Exp Zool 1974; 187:383-386[CrossRef][Medline]
  17. Downs SM, Daniel SAJ, Bornslaeger EA, Hoppe PC, Eppig JJ. Maintenance of meiotic arrest in mouse oocytes by purines: modulation of cAMP levels and cAMP phosphodiesterase activity. Gamete Res 1989; 23:323-334[CrossRef][Medline]
  18. Tsafriri A, Chun SY, Zhang R, Hsueh AJ, Conti M. Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors. Dev Biol 1996; 178:393-402[CrossRef][Medline]
  19. Schultz RM, Montgomery RR, Belanoff JR. Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis. Dev Biol 1983; 97:264-273[CrossRef][Medline]
  20. Bornslaeger EA, Poueymirou WT, Mattei P, Schultz RM. Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes. Exp Cell Res 1986; 165:507-517[CrossRef][Medline]
  21. Endo Y, Kopf GS, Schultz RM. Stage-specific changes in protein phosphorylation accompanying meiotic maturation of mouse oocytes and fertilization of mouse eggs. J Exp Zool 1986; 239:401-409[CrossRef][Medline]
  22. Choi T, Aoki F, Mori M, Yamashita M, Nagahama Y, Kohmoto K. Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos. Development 1991; 113::789-795[Abstract]
  23. Verlhac MH, Kubiak JZ, Clarke HJ, Maro B. Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes. Development 1994; 120:1017-1025[Abstract]
  24. Verlhac MH, Kubiak JZ, Weber M, Geraud G, Colledge WH, Evans MJ, Maro B. Mos is required for MAP kinase activation and is involved in microtubule organization during meiotic maturation in the mouse. Development 1996; 122:815-822[Abstract]
  25. Murray A, Gronvald FC, Nielsen JK, Faarup P. Synthesis of FF-MAS from lithocholic acid. Bioorg Med Chem Lett 2000; 10:1067-1068[CrossRef][Medline]
  26. Hegele-Hartung C, Kuhnke J, Lessl M, Grondahl C, Ottesen J, Beier HM, Eisner S, Eichenlaub-Ritter U. Nuclear and cytoplasmic maturation of mouse oocytes after treatment with synthetic meiosis-activating sterol in vitro. Biol Reprod 1999; 61:1362-1372[Abstract/Free Full Text]
  27. Tornell J, Billig H, Hillensjo T. Resumption of rat oocyte meiosis is paralleled by a decrease in guanosine 3',5'-cyclic monophosphate (cGMP) and is inhibited by microinjection of cGMP. Acta Vet Scand 1990; 139:511-517
  28. Hubbard CJ, Terranova PF. Inhibitory action of cyclic guanosine 5'-phosphoric acid (GMP) on oocyte maturation: dependence on an intact cumulus. Biol Reprod 1982; 26:628-632[Abstract]
  29. Dundore RL, Clas DM, Wheeler LT, Habeeb PG, Bode DC, Buchholz RA, Silver PJ, Pagani ED. Zaprinast increases cyclic GMP levels in plasma and in aortic tissue of rats. Eur J Pharmacol 1993; 249:293-297[CrossRef][Medline]
  30. Ballantyne S, Daniel DL, Wickens M. A dependent pathway of cytoplasmic polyadenylation reactions linked to cell cycle control by c-mos and CDK1 activation. Mol Biol Cell 1997; 8:1633-1648[Abstract]
  31. Maale G, Stein G, Mans R. Effects of cordycepin and cordycepintriphosphate on polyadenylic and ribonucleic acid-synthesizing enzymes from eukaryotes. Nature 1975; 255:80-82[CrossRef][Medline]
  32. Moore CL, Skolnik-David H, Sharp PA. Analysis of RNA cleavage at the adenovirus-2 L3 polyadenylation site. EMBO J 1986; 5:1929-1938[Medline]
  33. Sheets MD, Stephenson P, Wickens MP. Products of in vitro cleavage and polyadenylation of simian virus 40 late pre-mRNAs. Mol Cell Biol 1987; 7:1518-1529[Abstract/Free Full Text]
  34. Abraham RT, Acquarone M, Andersen A, Asensi A, Belle R, Berger F, Bergounioux C, Brunn G, Buquet-Fagot C, Fagot D, et al Cellular effects of olomoucine, an inhibitor of cyclin-dependent kinases. Biol Cell 1995; 83:105–20
  35. Glab N, Labidi B, Qin LX, Trehin C, Bergounioux C, Meijer L. Olomoucine, an inhibitor of the cdc2/cdk2 kinases activity, blocks plant cells at the G1 to S and G2 to M cell cycle transitions. FEBS Lett 1994; 353:207-211[CrossRef][Medline]
  36. Vesely J,, Havlicek L, Strnad M, Blow JJ, Donella-Deana A, Pinna L, Letham DS, Kato J, Detivaud L, Leclerc S. Inhibition of cyclin-dependent kinases by purine analogues. Eur J Biochem 1994; 224:771-786[Medline]
  37. Coticchio G, Fleming S. Inhibition of phosphoinositide metabolism or chelation of intracellular calcium blocks FSH-induced but not spontaneous meiotic resumption in mouse oocytes. Dev Biol 1998; 203::201-209[CrossRef][Medline]
  38. Pomerance M, Schweighoffer F, Tocque B, Pierre M. Stimulation of mitogen-activated protein kinase by oncogenic Ras p21 in Xenopus oocytes. Requirement for Ras p21-GTPase-activating protein interaction. J Biol Chem 1992; 267:16155-16160[Abstract/Free Full Text]
  39. Fukuda M, Gotoh Y, Kosako H, Hattori S, Nishida E. Analysis of the Ras p21/mitogen-activated protein kinase signaling in vitro and in Xenopus oocytes. J Biol Chem 1994; 269:33097-33101[Abstract/Free Full Text]
  40. Kim R,, Rine J, Kim SH. Prenylation of mammalian Ras protein in Xenopus oocytes. Mol Cell Biol 1990; 10:5945–5949
  41. Luttrell LM, Hawes BE, van Biesen T, Luttrell DK, Lansing TJ, Lefkowitz RJ. Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gß{gamma} subunit-mediated activation of mitogen-activated protein kinases. J Biol Chem 1996; 271:19443-19450[Abstract/Free Full Text]
  42. Igishi T, Gutkind JS. Tyrosine kinases of the Src family participate in signaling to MAP kinase from both Gq and Gi-coupled receptors. Biochem Biophys Res Commun 1998; 244:5-10[CrossRef][Medline]
  43. Macphee CH, Reifsnyder DH, Moore TA, Lerea KM, Beavo JA. Phosphorylation results in activation of a cAMP phosphodiesterase in human platelets. J Biol Chem 1988; 263:10353-10358[Abstract/Free Full Text]
  44. Degerman E, Belfrage P, Manganiello VC. cGMP-inhibited phosphodiesterases (PDE3 gene family). Biochem Soc Trans 1996; 24:1010-1014[Medline]
  45. Wiersma A, Hirsch B, Tsafriri A, Hanssen RG, Vandekant M, Kloosterboer HJ, Conti M, Hsueh AJ. Phosphodiesterase-3 inhibitors suppress oocyte maturation and consequent pregnancy without affecting ovulation and cyclicity in rodents. J Clin Inv 1998; 102:532-537[Medline]
  46. Bornslaeger EA, Mattei P, Schultz RM. Involvement of cAMP-dependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation. Dev Biol 1986; 114:453-462[CrossRef][Medline]
  47. Osborn JC, Moor RM. Time-dependent effects of alpha-amanitin on nuclear maturation and protein synthesis in mammalian oocytes. J Embryol Exp Morphol 1983; 73:317-338[Medline]
  48. Misteli T, Warren G. Mitotic disassembly of the Golgi apparatus in vivo. J Cell Sci 1995; 108:2715-2727[Abstract]
  49. Inoue M, Naito K, Nakayama K, Sato E. Mitogen-activated protein kinase translocates into the germinal vesicle and induces germinal vesicle breakdown in pig oocytes. Biol Reprod 1998; 58:130-136[Abstract/Free Full Text]
  50. Fissore RA, He CL, Vande W. Potential role of mitogen-activated protein kinase during meiosis resumption in bovine oocytes. Biol Reprod 1996; 55:1261-1270[Abstract]
  51. Peter M, Sanghera JS, Pelech SL, Nigg EA. Mitogen-activated protein kinases phosphorylate nuclear lamins and display sequence specificity overlapping that of mitotic protein kinase p34cdc2. Eur J Biochem 1992; 205:287-294[Medline]
  52. Colledge WH, Carlton MB, Udy GB, Evans MJ. Disruption of c-mos causes parthenogenetic development of unfertilized mouse eggs. Nature 1994; 370:65-68[CrossRef][Medline]
  53. Hashimoto N, Watanabe N, Furuta Y, Tamemoto H, Sagata N, Yokoyama M, Okazaki K, Nagayoshi M, Takeda N, Ikawa Y. Parthenogenetic activation of oocytes in c-mos-deficient mice. Nature 1994; 370:68-71[CrossRef][Medline]
  54. Araki K, Naito K, Haraguchi S, Suzuki R, Yokoyama M, Inoue M, Aizawa S, Toyoda Y, Sato E. Meiotic abnormalities of c-mos knockout mouse oocytes: activation after first meiosis or entrance into third meiotic metaphase. Biol Reprod 1996; 55:1315-1324[Abstract]
  55. Hegele-Hartung C, Lessl M, Ottesen J, Grøndahl C. Oocyte maturation can be induced by a synthetic meiosis activating sterol (MAS) leading to improvement of IVF rate in mice. Hum Reprod Suppl 1998; 13:98
  56. Choi T, Rulong S, Resau J, Fukasawa K, Matten W, Kuriyama R, Mansour S, Ahn N, Vande Woude GF. Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I. Proc Natl Acad Sci U S A 1996; 93:4730-4735[Abstract/Free Full Text]
  57. Zhao X, Singh B, Batten BE. The role of c-mos proto-oncoprotein in mammalian meiotic maturation. Oncogene 1991; 6:43-49[Medline]
  58. Fukasawa K, Murakami MS, Blair DG, Kuriyama R, Hunt T, Fischinger P, Vande Woude GF. Similarities between somatic cells overexpressing the mos oncogene and oocytes during meiotic interphase. Cell Growth Differ 1994; 5:1093-1103[Abstract]
  59. Schlessinger J. How receptor tyrosine kinases activate Ras. Trends Biochem Sci 1993; 18:273-275[CrossRef][Medline]
  60. Gutkind JS. The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem 1998; 273:1839-1842[Free Full Text]
  61. Lopez-Ilasaca M, Crespo P, Pellici PG, Gutkind JS, Wetzker R. Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI 3-kinase gamma. Science 1997; 275:394-397[Abstract/Free Full Text]
  62. McGeady P, Kuroda S, Shimizu K, Takai Y, Gelb MH. The farnesyl group of H-Ras facilitates the activation of a soluble upstream activator of mitogen-activated protein kinase. J Biol Chem 1995; 270::26347-26351[Abstract/Free Full Text]
  63. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem 1996; 271:695-701[Abstract/Free Full Text]
  64. Burns F, Rodger IW, Pyne NJ. The catalytic subunit of protein kinase A triggers activation of the type V cyclic GMP-specific phosphodiesterase from guinea-pig lung. Biochem J 1992; 283:487-491
  65. Smith RJ, Justen JM, McNab AR, Rosenbloom CL, Steele AN, Detmers PA, Anderson DC, Manning AM. U-73122: a potent inhibitor of human polymorphonuclear neutrophil adhesion on biological surfaces and adhesion-related effector functions. J Pharmacol Exp Ther 1996; 278:320-329[Abstract/Free Full Text]
  66. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J Biol Chem 1995; 270:27489-27494[Abstract/Free Full Text]
  67. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A 1995; 92:7686-7689[Abstract/Free Full Text]



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