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Endocrinology Vol. 143, No. 6 2221-2232
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Mitogen-Activated Protein Kinase Activity in Cumulus Cells Is Essential for Gonadotropin-Induced Oocyte Meiotic Resumption and Cumulus Expansion in the Mouse

You-Qiang Su, Karen Wigglesworth, Frank L. Pendola, Marilyn J. O’Brien and John J. Eppig

The Jackson Laboratory, Bar Harbor, Maine 04609

Address all correspondence and requests for reprints to: Dr. J. J. Eppig, The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609. E-mail: . jje{at}jax.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study investigated the participation of MAPK in the resumption of meiosis [germinal vesicle breakdown (GVB)] in oocytes and cumulus expansion using oocyte-cumulus cell complexes (OCC) from Mos-null mice (Mostm1Ev/Mostm1Ev, hereafter Mos-/-). MAPK activity was not detected in Mos-/- oocytes whether they matured in vivo or in vitro, with or without gonadotropin stimulation. Therefore, there are no pathways independent of MOS that activate MAPK during gonadotropin-induced maturation. In contrast, MAPK activity was always detected coincident with GVB in Mos+/+ oocytes. Moreover, MAPK activity was detected in cumulus cells before gonadotropin-induced GVB in OCC regardless of genotype. A specific inhibitor (U0126) of MEK, a MAPKK required for MAPK activity, inhibited gonadotropin-induced GVB in OCC of both Mos+/+ and Mos-/- mice. Activation of MAPK was downstream of elevation of cAMP. U0126 also inhibited cumulus expansion stimulated by FSH, epidermal growth factor, 8-bromo-cAMP, and recombinant growth differentiation factor-9. It is concluded that under the in vitro conditions used here, gonadotropin-induced GVB requires the participation of MAPK activity in the cumulus cells, but not in the oocyte. Moreover, the induction of cumulus expansion also requires the participation of MAPK, and this action is downstream of both elevation of cAMP and growth differentiation factor-9.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FULLY GROWN mammalian oocytes are maintained in ovarian follicles at the diplotene stage of the first meiotic prophase, commonly known as the germinal vesicle (GV) stage, by meiosis-arresting factors such as cAMP and/or purines such as hypoxanthine (1, 2, 3, 4, 5, 6, 7). The resumption of meiosis (G2-M transition), recognized morphologically by germinal vesicle breakdown (GVB), is triggered in healthy follicles by the preovulatory LH surge. LH promotes the production of a meiosis-inducing signal by granulosa cells (8).

Multiple signaling pathways probably participate in the resumption of meiosis, including pathways requiring MAPK. The activation of MAPK occurs via a complex cascade of kinases that include, immediately upstream, MAPK kinases (MAPKK, also called MEK), which are, in turn, phosphorylated by one of the MAPKK kinases (MAPKKK) such as MOS or RAF (9). MAPK in somatic cells can be activated rapidly in response to numerous extracellular stimuli. Thus, the MAPK pathway is able to transduce various extracellular signals to their intracellular targets and regulate cell cycle progression. The target proteins of MAPK are numerous and range from cytosolic, for example, cytoskeletal proteins and lamins, to nuclear, e.g. transcriptional activators (10, 11, 12).

Mammalian oocytes contain two isoforms of MAPK, ERK1 and ERK2, that are activated during in vitro meiotic maturation and are involved in regulating the normal progression of meiosis (13). However, the role of MAPK in regulating oocyte meiotic resumption is not fully resolved. MAPK in mouse, rat, and goat oocytes is activated after GVB during spontaneous gonadotropin-independent maturation in vitro (14, 15, 16, 17). Moreover, no MAPK activity is detected in oocytes from mice carrying a Mos-null mutation (Mostm1Ev/Mostm1Ev, hereafter Mos-/-) during spontaneous meiotic maturation, and GVB occurs normally both in vivo and in vitro in Mos-/- oocytes (18, 19, 20, 21, 22, 23). This evidence suggests that MAPK activity in the oocyte is not essential for meiotic resumption. In contrast, during equine, porcine, and bovine oocyte spontaneous maturation, MAPK is activated in the oocyte before GVB (24, 25, 26). Furthermore, in some special circumstances, MAPK is activated in mouse oocytes before GVB, for instance, when GVB is induced by okadaic acid or by MEK or MOS RNA injection (27, 28, 29). These reports suggest that MAPK may play a role in promoting GVB. Therefore, the role of MAPK activity within the oocyte in regulating meiotic resumption in mammals is still not well defined.

It is important to note that in the studies described above, spontaneous, hormone-independent, in vitro maturation systems were used to assess the possible function of MAPK in promoting GVB. Denuded oocytes (DO) or oocyte-cumulus cell complexes (OCC) isolated from large antral follicles undergo GVB in vitro without hormonal stimulation (30, 31). This experimental paradigm is quite different from LH-induced GVB in vivo, which is probably mediated by signals produced in follicular granulosa cells upon stimulation by LH (8, 32). Therefore, biochemical pathways required for gonadotropin-induced GVB in vivo might differ from those participating in spontaneous GVB in vitro (33, 34, 35). A useful in vitro model for assessing potential mechanisms of gonadotropin-induced GVB uses isolated OCC maintained in meiotic arrest with the purine hypoxanthine. In this case, FSH or epidermal growth factor (EGF) reverses the GVB-inhibiting effects of hypoxanthine via the production of a GVB-promoting signal by the cumulus cells (36). Using this model system it was found that inhibition of MAPK prevented FSH-induced, but not spontaneous, GVB (34, 35). However, as active forms of MAPK were detected in both the cumulus cell and the oocyte during FSH-induced GVB (35), it was not possible to determine whether MAPK activity in the oocyte, cumulus cells, or both was essential for GVB. If MAPK activity in the oocytes is essential for GVB in this gonadotropin-dependent system in vitro, it is possible that MAPK activity in oocytes is also essential for gonadotropin-induced maturation in vivo. If so, then MAPK must be induced by a MOS-independent pathway in vivo. If not, then OCC of Mos-/- females could be used to provide definitive information on the cellular localization of the MAPK pathway essential for gonadotropin-induced resumption of meiosis. The results of these studies are reported here.

Another physiological process occurring after the preovulatory LH surge and coincident with the resumption of meiosis is cumulus expansion. During this process, cumulus cells become embedded in a mucinous matrix containing hyaluronic acid (HA) synthesized by the cumulus cells in response to gonadotropin stimulation. Cumulus expansion also occurs in vitro when mouse OCC are stimulated with FSH or EGF (37, 38). However, in the mouse, HA synthesis and cumulus expansion fail to occur in the absence of the oocyte despite FSH stimulation. Coculture of oocytes with cumulus cells enables FSH-induced cumulus expansion to occur, indicating that an oocyte-secreted paracrine factor is required for cumulus expansion in the mouse (39, 40). This paracrine factor is probably the oocyte-specific growth differentiation factor-9 (GDF-9), because recombinant GDF-9 (GDF-9) can promote Has2 (a key gene for HA synthesis) expression and cumulus expansion (41). However, little is known about the biochemical pathways involved in the induction of cumulus expansion, especially those induced by GDF-9. Hence, in this study the question of whether a MAPK-dependent pathway is essential for gonadotropin- and GDF-9-induced cumulus expansion was addressed. We assessed the effect of a specific MEK inhibitor, U0126, on cumulus expansion induced by FSH, EGF, 8-bromo-cAMP, or GDF-9 using intact OCC or oocytectomized (OOX) cumulus complexes from Mos-/- mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Mos mutant mice, originally produced by Dr. Martin J. Evans (20), were raised in the research colony of the investigators at The Jackson Laboratory (Bar Harbor, ME). Offspring produced by mating Mos+/- females with either Mos+/- or Mos-/- males were genotyped to identify Mos-/- or Mos+/+ females for experiments by PCR using specific Mos and Neo primer pairs as previously described (42).

Oocyte isolation and culture
Immature 22- to 24-d-old mice were used for all experiments. Follicle development was stimulated by ip injection of each mouse with 5 IU PMSG (National Hormone and Peptide Program, NIDDK). OCC were isolated 44–48 h post-PMSG injection as described previously (43) and were collected in medium containing 4 mM hypoxanthine (hereafter HX-medium) to maintain oocytes at the GV stage. The culture medium used for all experiments was bicarbonate-buffered MEM{alpha} (Life Technologies, Inc., Grand Island, NY) with Earle’s salts, supplemented with 75 mg/liter penicillin G, 50 mg/liter streptomycin sulfate, 0.23 mM pyruvate, and 3 mg/ml crystallized lyophilized BSA. All medium components were purchased from Sigma (St. Louis, MO). After being washed through three additional changes of medium, OCC were allocated and cultured in four-well plastic culture dishes (Nunclon, Nunc, Copenhagen, Denmark) containing 400 µl of the appropriate culture medium at 37 C in a modular incubation chamber (Billups Rothenberg, Del Mar, CA) infused with 5% O2, 5% CO2, and 90% N2.

MAPK activity in oocytes and cumulus cells
To examine the kinetics of gonadotropin-induced oocyte meiotic resumption and MAPK activation in both oocytes and cumulus cells in vivo, Mos-/- or Mos+/+ female mice were injected with hCG (5 IU/mouse; Sigma) or, as a control group, with the same amount of saline after being primed with PMSG for 48 h. Intact OCC were then isolated at 0.5–16 h postinjection, and the incidence of oocyte GVB was scored under a stereomicroscope after cumulus cells were stripped from the oocyte by repeated pipetting of the OCC through a fine-bore pipette. After the removal of DO, cumulus cells were collected by centrifugation at 4 C at 14,000 rpm. Samples of oocytes and cumulus cells for Western blot analysis were collected and stored at -70 C until use. To examine the kinetics of gonadotropin-induced GVB and MAPK activation in both oocytes and cumulus cells in vitro, isolated Mos-/- or Mos+/+ OCC were cultured in HX-medium with or without 100 IU/liter human recombinant FSH (hFSH; National Hormone and Pituitary Program, NIDDK) for 20 h. During the culture the incidence of GVB was scored at 1–20 h of culture, and the samples of oocytes and cumulus cells were then collected and stored at -70 C until MAPK assay. MAPK activity was detected as described below by Western blot analysis using an anti-active MAPK antibody, which specifically identifies the active phosphorylated forms of MAPK.

Electrophoresis and Western blot analysis
Proteins from 30 DO or cumulus cells from 30 OCC were extracted using 2x Laemmli sample buffer (44). The lysates were heated to 100 C for 4 min, then cooled on ice for 4 min and centrifuged at 14,000 rpm for 5 min. Western blot analysis was then performed as described previously (35). The phosphorylated forms of MAPK were detected using a monoclonal anti-MAPK, activated (diphosphorylated ERK1 and -2) antibody (Sigma). The second antibody used for detection of MAPK-primary antibody complex was horseradish peroxidase-conjugated goat antimouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Proteins on the membrane were visualized using the ECL detection system (Pierce Chemical Co., Rockford, IL). After the initial analysis, the membranes were washed in a stripping buffer (Pierce Chemical Co.) to remove bound antibodies and were reprobed with a polyclonal anti-MAPK antibody (Sigma) to detect the total amount of MAPK (phosphorylated and unphosphorylated forms). The second antibody used for the reprobing was horseradish peroxidase-conjugated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.).

Oocytectomy
The microsurgical removal of the oocyte from OCC was performed using a microsurgical protocol described previously (39). Briefly, each complex was held with the holding pipette using negative pressure. A lancing pipette was pushed through the complex and into the holding pipette. Upon withdrawal of the lancing pipette, the negative pressure in the holding pipette aspirated most or all of the oocyte. The resulting OOX complexes consisted of the spherical zona pellucida surrounded by the cumulus cell mass, but deprived of the oocyte.

In vitro treatments
Mos-/- or Mos+/+ OCC were cultured in HX-medium with or without factors to induce GVB: 100 IU/liter hFSH or 10 ng/ml EGF (Collaborative Biomedical Products, Bedford, MA) for 17–18 h. Alternatively, GVB was induced by briefly exposing the complexes for 3 h to 1 mM 8-bromo-5'-cAMP (8-Br-cAMP; Sigma), then washed and transferred to fresh HX-medium and cultured for an additional 14–15 h. In experiments where the spontaneous maturation model was used, Mos-/- or Mos+/+ OCC were cultured in medium without HX for 14 h. To investigate the role of MAPK in ligand-induced and spontaneous meiotic maturation, the highly specific MEK inhibitor, U0126 (10 µM), or its inactive analog, U0124 (Calbiochem, La Jolla, CA), was added to the culture medium at the beginning of culture. In cumulus expansion experiments, OOC or OOX cumulus complexes were cultured for 15 h in 50-µl drops of medium supplemented with 5% FBS in Falcon petri dishes covered by equilibrated paraffin oil. To induce cumulus expansion in OCC, the complexes were treated with 100 IU/liter hFSH, 10 ng/ml EGF, or 1 mM 8-Br-cAMP. To induce cumulus expansion in OOX, the complexes were treated with 100 ng/ml GDF-9 or control medium (i.e. deficient in GDF-9, but containing 2% FCS). GDF-9 and its control medium were provided by Dr. Martin M. Matzuk (Baylor College of Medicine, Houston, TX). To test the involvement of MAPK in these processes, 10 µM U0126 or U0124 was added to the culture medium at the beginning of culture.

Evaluation of oocyte meiotic maturation and cumulus expansion
Oocytes were assessed for maturation at the end of the culture period by removing cumulus cells and scoring the oocytes for GVB (meiotic resumption) under a stereomicroscope. The degree of expansion was scored according to a subjective scale from 0 (no expansion) to 4 (complete expansion), and a cumulus expansion index was calculated (range, 0–4.00) as described in detail previously (45, 46).

Statistical analysis
Each oocyte maturation experiment was conducted at least 3 times independently with 30 oocytes/group/experiment, and the data are reported as the mean percentage of GVB ± SEM. The number of experimental replicates is indicated in the figure legends. All frequencies were subjected to arcsin transformation and statistically compared by ANOVA (except in some experiments, as indicated in the figure legend, paired t test was used) using StatView software (SAS Institute, Inc., Cary, NC). When a significant F ratio was defined by ANOVA, groups were compared using Fisher’s protected least significant difference post hoc test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of MAPK in oocytes during gonadotropin-induced meiotic maturation in vivo
Almost all Mos+/+ and Mos-/- oocytes underwent GVB by 4 h post-hCG injection (96.7± 2.1% in Mos+/+ oocytes and 93.8 ± 3.1% in Mos-/- oocytes, respectively; Fig. 1Go). In the control groups none of the oocytes isolated from Mos+/+ or Mos-/- mice underwent GVB at any time postsaline injection (data not shown), and no active forms of MAPK were detected at any time in either Mos+/+ or Mos-/- oocytes (Fig. 2Go, A and C). In Mos+/+ oocytes, activation of MAPK was detected at 4 h post-hCG injection, and the maximal activation was observed at 12–16 h post-hCG injection (Fig. 2BGo, top panel). The total amount of MAPK protein did not change at any time (Fig. 2BGo, bottom panel). Thus, MAPK was activated in Mos+/+ oocytes coincident with GVB in vivo. However, in Mos-/- oocytes, no active forms of MAPK were detected at any time before or after GVB during the process of hCG-induced meiotic maturation in vivo (Fig. 2DGo, top panel) despite the fact that similar amounts of MAPK protein detected in the oocyte (Fig. 2DGo, bottom panel).



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  		Figure 1. Kinetics of GVB in vivo. Mos+/+ and Mos-/- mice were injected with hCG (5 IU/mouse) after priming with PMSG for 48 h. OCC were isolated at various times after hCG injection, and the rate of GVB was scored. Experiments were repeated five times. Where there are no common letters over the points, the groups were significantly different (P < 0.05).

 


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  		Figure 2. Kinetics of MAPK activation within cumulus cells and oocytes in vivo. Mos+/+ and Mos-/- mice were injected with hCG (5 IU/mouse) or the same amount of saline after priming with PMSG for 48 h. OCC were isolated at various times post-hCG or -saline injection. Samples of oocytes or cumulus cells for MAPK assay were collected after GVB was scored. MAPK activity was assessed by Western blot analysis using a specific antiactive MAPK monoclonal antibody (indicated in the top panels). The total amounts of MAPK protein (indicated in the bottom panels) were determined by reprobing the same blot using an anti-MAPK polyclonal antibody. A total of 30 oocytes or cumulus cells from the same 30 OCC were loaded into each lane. The positions of the two MAPK isoforms (ERK1 and -2) are indicated on the left of each gel. The same treatments were applied in all of the following Western blot figures. Each experiment was repeated three times with similar results, and a representative gel is shown. A, Blots of cumulus cells and oocytes isolated from saline-injected Mos+/+ mice. B, Blots of cumulus cells and oocytes isolated from hCG-injected Mos+/+ mice. C, Blots of cumulus cells and oocytes isolated from saline-injected Mos-/- mice. D, Blots of cumulus cells and oocytes isolated from hCG-injected Mos-/- mice.

 
Activation of MAPK in oocytes during gonadotropin-induced meiotic maturation in vitro
hFSH induced GVB after 12 h of culture (35.3 ± 3% and 34.6 ± 8.3% in Mos+/+ and Mos-/- OCC, respectively) compared with oocytes cultured in medium without FSH (4.5 ± 1.21% and 14.3 ± 4.6% in Mos+/+ and Mos-/- OCC, respectively; Fig. 3Go). In the control groups without hFSH treatment, no active forms of MAPK were detected in Mos+/+ (Fig. 4AGo) or Mos-/- (Fig. 4CGo) oocytes. During FSH-induced meiotic resumption in vitro, a low level of MAPK activation was detected in Mos+/+ oocytes at 4–8 h of FSH stimulation, and greater activation was observed at 12–20 h of FSH stimulation (Fig. 4BGo). Thus, during gonadotropin-induced maturation of Mos+/+ oocytes in vitro, MAPK was activated at about the time of GVB. However, under the same culture conditions, no active forms of MAPK were detected in Mos-/- oocytes at any time before or after GVB (Fig. 4DGo, top panel) despite the presence of similar amounts of MAPK protein (Fig. 4DGo, bottom panel).



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  		Figure 3. Kinetics of GVB in vitro. Mos+/+ (A) and Mos-/- (B) OCC were cultured in HX-medium and treated, or not, with 100 IU/liter hFSH (HX+FSH or HX, respectively) for 20 h in vitro. The rate of GVB was scored at various time points during the culture. Each experiment was repeated three times. The differences between HX+FSH and HX groups at the same time points were statistically compared by t test. *, P < 0.05; **, P < 0.01.

 


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  		Figure 4. Kinetics of MAPK activation within cumulus cells and oocytes in vitro. Mouse OCC were cultured in HX-medium and treated, or not, with 100 IU/liter hFSH for 20 h in vitro. During culture, after GVB was scored at various time points, samples of cumulus cells and oocytes were collected for MAPK activity assay. A total of 30 oocytes or cumulus cells from the same 30 OCC were loaded into each lane. Each experiment was repeated 3 times with similar results, and a representative gel is shown. A, Blots of cumulus cells and oocytes from Mos+/+ OCC cultured in HX-medium without hFSH treatment. B, Blots of cumulus cells and oocytes from Mos+/+ OCC cultured in HX-medium with 100 IU/liter hFSH treatment. C, Blots of cumulus cells and oocytes from Mos-/- OCC cultured in HX-medium without hFSH treatment. D, Blots of cumulus cells and oocytes from Mos-/- OCC cultured in HX-medium with 100 IU/liter hFSH treatment.

 
Activation of MAPK in cumulus cells during gonadotropin-induced meiotic maturation in vivo and in vitro
During gonadotropin-induced meiotic maturation, activation of MAPK was detected in cumulus cells of both Mos+/+ and Mos-/- OCC, both in vitro and in vivo. In vivo, only low levels of active MAPK were detected in the cumulus cells in the control groups (Mos+/+, Fig. 2AGo; Mos-/-, Fig. 2CGo). However, activation of MAPK was detected at 0.5 h post-hCG injection in both Mos-/- and Mos+/+ cumulus cells, and maximal activation was observed at 2–4 h. MAPK activity decreased at 12 h, and active forms of MAPK were not detected at 16 h (Fig. 2Go, B and D). In vitro, only slight activation of MAPK was detected at all time points in the cumulus cells of the control groups (Mos+/+, Fig. 4AGo; Mos-/-, Fig. 4CGo). In contrast, during FSH-induced meiotic maturation in both Mos+/+ (Fig. 4BGo) and Mos-/- (Fig. 4DGo) OCC in vitro, activation of MAPK in cumulus cells was detected at 1 h of FSH stimulation. Maximal levels were observed at 4–8 h; MAPK activity in cumulus cells decreased at 12 h of FSH stimulation, and active forms of MAPK were greatly reduced by 20 h. Both in vitro and in vivo, changes in the active forms of MAPK were not due to changes in the amounts of MAPK protein (Fig. 4Go, B and D, and Fig. 2Go, B and D, bottom panels). Thus, MAPK activity in cumulus was elevated before GVB in both Mos+/+ and Mos-/- OCC during in vitro and in vivo gonadotropin-induced meiotic maturation.

Inhibition of MAPK activation in Mos-/- cumulus cells blocked hormone-induced, but not spontaneous, meiotic resumption in vitro
The effects of inhibition of MAPK activation in the cumulus cell on FSH-induced and spontaneous GVB in vitro were determined using Mos-/- OCC, because MAPK activity in the oocyte was already absent. As shown in Fig. 5AGo, when these OCC were cultured in HX-medium for 17–18 h, only 8.4% of the oocytes underwent GVB. When stimulated with 100 IU/liter hFSH, the percentage of GVB increased to 34.4%. The specific MEK inhibitor U0126 (10 µM) reduced the stimulatory effect of hFSH to 9.9% GVB. In contrast, 10 µM U0124, an inactive analog of U0126, did not block the stimulatory effect of FSH (33.0% GVB).



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  		Figure 5. Effects of MEK inhibition on Mos-/- oocyte meiotic resumption in vitro. A, Mos-/- OCC were cultured in HX-medium without any treatment (HX) or were cultured in HX-medium and treated with 100 IU/liter hFSH (HX+FSH), 100 IU/liter hFSH plus 10 µM U0126 (HX+FSH+U0126), or 100 IU/liter hFSH plus 10 µM U0124 (HX+FSH+U0124) for 17–18 h in vitro. The incidence of GVB was scored at the end of culture. B, Mos-/- OCC were cultured in maturation medium and allowed to mature spontaneously (control) or were cultured in maturation medium and treated with 10 µM U0126 or 10 µM U0124 for 14 h. The incidence of GVB was scored at the end of culture. Each experiment was repeated six times. Where there are no common letters over the bars, the groups were significantly different (P < 0.05).

 
When Mos-/- OCC were cultured in maturation medium without HX for 14 h, all oocytes underwent spontaneous GVB (Fig. 5BGo). However, unlike Mos+/+ oocytes, which arrested at the metaphase II stage with normal-appearing first polar bodies after a 14-h culture (Fig. 6AGo), Mos-/- oocytes showed abnormal meiotic progression at this time (Fig. 6DGo). Oocytes were assigned to one of three classes according to the criteria described by Hirao and Eppig (42): class I included oocytes with up to two polar bodies; class II oocytes exhibited an extremely large polar body or underwent fragmentation; and class III had the appearance of two-cell embryos. In contrast to its inhibitory effect on FSH-induced GVB, U0126 (10 µM) did not block spontaneous GVB of Mos-/- OCC. Similar effects of U0126 on FSH-induced and spontaneous GVB were observed in Mos+/+ OCC (data not shown). However, Mos+/+ oocytes, when treated with 10 µM U0126 and cultured for 14 h, showed phenotypes similar to those of Mos-/- oocytes cultured for 14 h in maturation medium (Fig. 6CGo). Mos+/+ oocytes treated with 10 µM U0124 had normal-appearing PB1 (Fig. 6BGo).



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  		Figure 6. Photomicrographs of Mos+/+ and Mos-/- oocytes. OCC were cultured in maturation medium treated with or without 10 µM U0126 or U0124 for 14 h. At the end of culture, OCC were denuded, and meiotic progression was scored. A, Mos+/+ oocytes progressed to metaphase II stage with normal-appearing first polar bodies. B, Mos+/+ oocytes treated with U0124 progressed to metaphase II stage with normal-appearing first polar bodies. C, Mos+/+ oocytes treated with U0126 showed the same abnormal meiotic phenotypes as Mos-/-oocytes. D, Mos-/- oocytes with abnormal meiotic phenotypes. Closed arrows indicate oocytes that produced two polar bodies; closed arrowheads indicate oocytes that cleaved to the two-cell stage; open arrows indicate oocytes with an extremely large polar body; open arrowheads indicate oocytes underwent fragmentation. Scale bar, approximately 100 µm.

 
HX-maintained meiotic arrest in mouse OCC can also be reversed by other ligands, such as EGF, through a mechanism mediated by interaction with cumulus cells (36). Therefore, the involvement of MAPK activity in the response of cumulus cells treated with EGF was also determined. As shown in Fig. 7Go, EGF promoted GVB in 44.6% of Mos-/- cumulus cell-enclosed oocytes, comparable to FSH-induced maturation. The stimulatory effect of EGF was blocked by 10 µM U0126, but not U0124, supporting the involvement of MAPK activity within the cumulus cell in EGF-induced meiotic resumption.



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  		Figure 7. Effect of MEK inhibition on EGF- or 8-Br-cAMP-induced GVB in Mos-/- oocytes. Mos-/- OCC were cultured in HX-medium (HX), were cultured in HX medium and treated with 10 ng/ml EGF (indicated as EGF) for 17–18 h, or were pulsed with 1 mM 8-Br-cAMP (indicated as 8-Br-cAMP) for 3 h, then transferred into fresh HX-medium for another 14–15 h. At the beginning of each culture, OCC were treated with or without 10 µM U0126 or U0124. The incidence of GVB was scored at the end of culture. Each experiment was repeated three times. Where there are no common letters over the bars, the groups were significantly different (P < 0.05).

 
FSH and EGF-induced reversal of hypoxanthine-mediated meiotic arrest in isolated cumulus cell-enclosed oocytes can be mimicked by pulsing meiotic-arrested mouse cumulus-enclosed oocytes with a high concentration of cAMP analog (36, 47, 48). This finding suggested that the cAMP-dependent pathway is involved in the induction of meiotic resumption. However, it is not clear whether the MAPK-dependent step occurs before or after the activation of PKA during the process of meiotic induction. Therefore, the involvement of MAPK in cAMP-induced reversal of hypoxanthine-mediated meiotic arrest was determined. Mos-/- OCC were exposed to a 3-h pulse of 1 mM 8-Br-cAMP and subsequently transferred to 8-Br-cAMP-free medium in the continued presence of HX. As shown in Fig. 7, 8-Br-cAMP promoted GVB in Mos-/- cumulus cell- enclosed oocytes at a rate of 48.5%, comparable to the incidence of GVB induced by FSH or EGF. The stimulatory effect of 8-Br-cAMP was blocked by 10 µM U0126, but not by U0124, suggesting the MAPK activation occurs downstream of the activation of PKA.

To determine whether the inhibition of ligand-induced GVB by the MEK inhibitor in Mos-/- OCC is attributable to its inhibition of MAPK activity in Mos-/- cumulus cells, MAPK activity was assessed within cumulus cells of Mos-/- OCC during FSH-, EGF-, and 8-Br-cAMP-induced meiotic resumption in the presence or absence of U0126 and U0124. As shown in Fig. 8Go, when Mos-/- OCC were treated with hFSH (100 IU/liter), EGF (10 ng/ml), or 8-Br-cAMP (1 mM) for 4 h, significant activation of MAPK was detected in cumulus cells. This activation was inhibited by 10 µM U0126, but not U0124, thus indicating that MAPK activity in the cumulus cell participates in ligand-induced meiotic resumption.



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  		Figure 8. Effect of MEK inhibition on hormone-induced MAPK activation in cumulus cells of Mos-/- OCC. OCC were cultured in HX-medium and treated with 100 IU/liter hFSH, 10 ng/ml EGF, or 1 mM 8-Br-cAMP for 4 h. At the beginning of each culture, OCC were treated, or not, with 10 µM U0126 or U0124. Cumulus cells from 30 OCC were isolated for MAPK activity assay at the end of culture. Each experiment was repeated three times with similar results, and a representative gel is shown. Lane 1, Cumulus cells isolated from OCC cultured in HX-medium alone; lane 2, cumulus cells isolated from OCC cultured in HX-medium and treated with 100 IU/liter hFSH; lane 3, cumulus cells isolated from OCC cultured in HX-medium and treated with 100 IU/liter hFSH plus 10 µM U0126; lane 4, cumulus cells isolated from OCC cultured in HX-medium and treated with 100 IU/liter hFSH plus 10 µM U0124; lane 5, cumulus cells isolated from OCC cultured in HX-medium and treated with 10 ng/ml EGF; lane 6, cumulus cells isolated from OCC cultured in HX-medium and treated with 10 ng/ml EGF plus 10 µM U0126; lane 7, cumulus cells isolated from OCC cultured in HX-medium and treated with 10 ng/ml EGF plus 10 µM U0124; lane 8, cumulus cells isolated from OCC cultured in HX-medium and treated with 1 mM 8-Br-cAMP; lane 9, cumulus cells isolated from OCC cultured in HX-medium and treated with 1 mM 8-Br-cAMP plus 10 µM U0126; lane 10, cumulus cells isolated from OCC cultured in HX-medium and treated with 1 mM 8-Br-cAMP plus 10 µM U0124.

 
Participation of MAPK activity within the Mos-/- cumulus cell in the process of cumulus expansion
In addition to oocyte maturation, the expansion or mucification of the cumulus oophorus also occurs in response to the preovulatory LH surge in vivo and to treatment with FSH, EGF, or cAMP in vitro (38, 49, 50). Therefore, the participation of MAPK-dependent pathways in ligand-induced cumulus expansion in vitro was investigated using Mos-/- OCC. As shown in Table 1Go, hFSH (100 IU/liter), EGF (10 ng/ml), and 8-Br-cAMP (1 mM) induced cumulus expansion of Mos-/- OCC to the maximal degree, with cumulus expansion indexes (CEIs) of 3.99, 4, and 3.95, respectively. Cumulus expansion induced by all of these treatments was inhibited by 10 µM U0126, but not by U0124. Thus, MAPK activity in the cumulus cells is involved in ligand-induced cumulus expansion in vitro, and this MAPK-dependent step occurs downstream of the activation of PKA.


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  		Table 1. Effect of MAPKK inhibitor on hormone-induced cumulus expansion in Mos-/- OCC

 
As cumulus expansion in the mouse requires the presence of an oocyte-secreted enabling factor, it was important to determine whether the participation of MAPK within the cumulus cells in promoting cumulus expansion takes place downstream of the action of this enabling factor. The cumulus expansion-enabling factor is probably GDF-9, as this oocyte-specific paracrine factor can promote cumulus expansion in the absence of the oocyte (41). Therefore, the effect of the MEK inhibitor on GDF-9-induced cumulus expansion in OOX complexes was determined. As shown in Table 2Go, as expected, no cumulus expansion was detected in control group, the control medium-treated group, or the group treated with FSH. In these groups cumulus cells attached to the substratum and assumed a fibroblastic appearance (Fig. 9Go, A–C). In contrast, GDF-9 at a concentration of 100 ng/ml induced the expansion of OOX cumulus complexes, with a CEI of 3.79. Cumulus cells in the GDF-9-treated group maintained a spherical appearance and expanded into a three-dimensional, gelatinous sphere (Fig. 9DGo). The stimulatory effect of GDF-9 was almost totally inhibited by treatment with 10 µM U0126, with a CEI of 0.29, and cumulus cells were similar in appearance to those in the control group (Fig. 9EGo). Treatment with 10 µM U0124 (the inactive form) had no inhibitory effect on GDF-9-induced cumulus expansion, with a CEI of 3.94, and cumulus cells appeared the same as in the GDF-9-treated group (Fig. 9FGo). Therefore, the cumulus expansion-promoting activity of GDF-9 requires MAPK activity in the cumulus cells.


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  		Table 2. Effect of MAPKK inhibitor on GDF-9-induced cumulus expansion in OOX

 


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  		Figure 9. Effect of MEK inhibition on GDF-9-induced cumulus expansion in Mos-/- OOX cumulus complexes. Mos-/- OOX cumulus complexes were cultured in maturation medium supplemented with 5% FBS for 15 h. During the culture, different treatments were performed: A, control group without any treatments; B, OOX treated with control medium; C, OOX treated with 100 IU/liter hFSH; D, OOX treated with 100 ng/ml GDF-9; E, OOX treated with 100 ng/ml GDF-9 plus 10 µM U0126; F, OOX treated with 100 ng/ml GDF-9 plus 10 µM U0124. Scale bar, approximately 100 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results presented in this study demonstrate that MAPK is not activated in Mos-/- oocytes induced to mature by gonadotropin either in vitro or in vivo. In contrast, MAPK in the cumulus cell is activated before gonadotropin-induced GVB. Furthermore, inhibition of MAPK activation in the cumulus cells blocks both the induction of GVB and cumulus expansion of Mos-/- oocyte-cumulus complexes by FSH, EGF, and 8-Br-cAMP. Therefore, under the culture conditions used here, the activation of MAPK in cumulus cells, but not in the oocyte, participates in gonadotropin-induced meiotic resumption and cumulus expansion. The MAPK-dependent step(s) occurs downstream of the activation of PKA. Moreover, the requirement for MAPK in the induction of cumulus expansion also occurs downstream of the action of the probable cumulus expansion-enabling factor, GDF-9.

Activation of MAPK in the oocyte is not essential for gonadotropin-induced meiotic resumption
As GVB occurs during the spontaneous maturation of Mos-/- oocytes in vitro, and these oocytes do not express active MAPK, it was clear from previous studies that spontaneous GVB in the mouse does not require MAPK activity (18, 19, 22, 23). However, it was not known previously whether gonadotropin-induced maturation might require MAPK activation by a pathway independent of MOS function (51). Here, it is demonstrated that meiotic resumption (GVB) occurs apparently normally in Mos-/- oocytes during both in vitro FSH-induced and in vivo hCG-induced meiotic maturation. Yet, no active forms of MAPK were detected before or after GVB in these processes despite the presence of normal amounts of MAPK protein in Mos-/- oocytes. Therefore, there is no alternative, MOS-independent, mechanism to activate MAPK during gonadotropin-induced meiotic resumption in the mouse oocyte, and MAPK activity in the oocyte is not required for GVB in vivo or in vitro.

Treatment of Mos+/+ oocytes with a specific inhibitor of MEK produced the same phenotype as that of Mos-/- oocytes, i.e., the oocytes entered interphase instead of arresting at metaphase II. This supports previous findings that MAPK is required for maintaining metaphase II arrest in the oocyte (18, 19, 20, 21, 22, 23).

The involvement of MOS/MAPK in oocyte meiotic resumption has been well studied in amphibian oocytes, particularly in frog oocytes. As in mouse oocytes, MOS expression is restricted to oocytes in Xenopus and is required for the activation of MAPK (52). Earlier studies show that MOS/MAPK is essential for progesterone-induced GVB in Xenopus oocytes, thus indicating that triggering meiotic resumption might be a frog-specific MOS/MAPK function (53). However, recent studies show that suppression of MAPK activation in Xenopus oocytes does not inhibit progesterone-induced GVB, suggesting that activation of MAPK is actually not necessary for the resumption of meiosis (54, 55). Therefore, an alternative, yet universally similar, pathway might exist in both mouse and frog oocytes for triggering the resumption of meiosis (54).

MAPK activity in the cumulus cells is required for gonadotropin-induced GVB
That gonadotropin-induced GVB requires MAPK activity in cumulus cells, but not in the oocyte, is demonstrated by the following observations. First, MAPK in cumulus cells of Mos-/- and Mos+/+ OCC was activated by gonadotropins before GVB in vitro and in vivo. Second, inhibition of MAPK activity in Mos+/+ OCC blocked FSH-induced GVB in vitro. Third, inhibition of MAPK activity did not block spontaneous, gonadotropin-independent GVB. These observations are consistent with the other findings in normal mouse OCC (34, 35). Fourth, inhibition of MAPK activity in Mos-/- OCC blocked FSH-induced GVB in vitro. As the oocyte in these OCC does not express active MAPK even in the absence of the MEK inhibitor, blocking GVB in these OCC by the MEK inhibitor must target MAPK activity induced by gonadotropin in the cumulus cells.

FSH-induced reversal of hypoxanthine-mediated meiotic arrest in mouse OCC in vitro is a useful experimental model for examining the potential biochemical pathways that participate in gonadotropin-induced GVB. It is a relatively simple system involving only two cell types, cumulus cells and the oocyte. However, because mouse cumulus cells do not express LH receptors, GVB is not induced by LH, but by FSH and EGF. Moreover, the time of GVB is significantly delayed compared with that of LH-induced GVB in vivo. Nevertheless, the sequence of FSH-stimulated MAPK activation in the cumulus cells and the time of GVB in vitro are essentially the same as those observed during gonadotropin-induced maturation in vivo. The cell to cell interactions involved in gonadotropin-induced maturation in vivo are complex and poorly understood. Clearly, a variety of experimental approaches will be required to understand them, and no single system will be adequate.

Participation of MAPK activity in the cumulus cells in promoting cumulus expansion
The MAPK-dependent signaling pathway may participate in several gonadotropin- and growth factor-mediated processes in granulosa cells. For example, steroidogenesis in isolated ovarian granulosa cells or immortalized granulosa cell lines appears dependent upon gonadotropin (or EGF) stimulation of MAPK activity (56, 57, 58, 59, 60, 61). In addition to promoting oocyte maturation, another physiological response of cumulus cells to ligand stimulation is cumulus expansion, which occurs concomitantly with oocyte maturation. Cumulus expansion, also called cumulus mucification, is a process that occurs after the preovulatory gonadotropin surge and requires the synthesis and secretion of HA by cumulus cells. The secreted HA disperses the cumulus cells and embeds them in a mucus-like matrix (37, 50, 62). In vitro, cumulus expansion occurs when isolated OCC are treated with FSH, EGF, or a cAMP analog, such as 8-Br-cAMP (37, 38, 39, 47, 50, 63, 64, 65). Because cumulus expansion is an obvious morphological response of cumulus cells in OCC to FSH stimulation, it was used in the present study to assess a possible role of MAPK activation in cumulus cells during FSH-induced meiotic maturation in vitro. It was found that U0126, but not U0124, blocked FSH-, EGF-, and 8-Br-cAMP-induced cumulus expansion in Mos-/- OCC, suggesting that MAPK activity within the cumulus cells is required for the induction of cumulus expansion.

Cumulus expansion requires the participation of an oocyte-secreted cumulus expansion-enabling factor (39, 40, 66, 67). GDF-9, a member of the secreted TGFß superfamily, could substitute for oocytes and oocyte-conditioned medium to stimulate cumulus expansion in OOX cumulus complexes (41). Therefore, GDF-9 is probably an oocyte-secreted, cumulus expansion-enabling factor. Here we assessed whether the MAPK-dependent steps in promoting cumulus expansion occur downstream of the effects of GDF-9. To resolve this, the effect of MEK inhibitor on GDF-9-induced cumulus expansion in OOX cumulus complexes was tested. It was observed that MAPK activity is required for GDF-9 to promote cumulus expansion, indicating that the required MAPK-dependent steps in the induction of cumulus expansion occur downstream of GDF-9 stimulation.

Members of the TGFß superfamily bind to a distinctive combination of type II and type I serine/threonine kinase receptors and transduce signals through phosphorylation of specific SMAD proteins to alter transcription (68). The MAPK pathway is known to cross-talk with members of TGFß family ligands and their SMAD intermediaries. In some cases this interaction is antagonistic, whereas in others it is agonistic (69). As shown here, MAPK activity is required for GDF-9 to promote cumulus expansion, indicating that the MEK-MAPK pathway interacts with the GDF-9 signal transduction cascade. However, as neither receptors nor signaling pathways for GDF-9 have been clearly identified, it is not clear at which step the cross-talk occurs. The MEK-MAPK signaling pathway may directly transduce the GDF-9 stimulus in the cumulus cells.

PKA can regulate MAPK activity in many types of cells; both inhibitory and stimulatory effects have been described. For example, in Rat lhER fibroblasts, PKA suppresses the activation of MAPK by inhibiting RAF-1 (70). In contrast, PKA promotes the activation of MAPK in PC12 cells by stimulating B-RAF (71). In the present study, 8-Br-cAMP increased MAPK activity in cumulus cells. Moreover, inhibition of MAPK activity blocked both 8-Br-cAMP-induced oocyte GVB and cumulus expansion. Thus, the cAMP-PKA pathway regulates these functions of cumulus cells by activation of MAPK. These results are consistent with the finding in granulosa cells that cAMP-PKA activates MAPK (56, 57, 60). The mechanisms underlying cAMP-PKA activation of MAPK in cumulus cells are not clear. For example, MAPK activation could be many steps downstream from immediate PKA substrate phosphorylation.

It is not known whether the two gonadotropin-induced functions of cumulus cells investigated here, the induction of GVB and cumulus expansion, are related. Cumulus expansion and oocyte maturation are both accompanied by disassembly of gap junctions (72, 73, 74). Disassembly of gap junctional communication would certainly disrupt the transfer of meiosis-arresting factors, such as cAMP, that are essential for the maintenance of oocyte meiotic arrest (74, 75). However, the induction of GVB probably involves gonadotropin-induced production of a positive maturation-inducing signal by the granulosa cells as well as termination of the flow of meiosis-arresting factors (32). Further investigation will be necessary to determine whether all ligand-induced functions of cumulus cells require an active MAPK pathway or whether only functions that are either functionally or temporally linked require this signaling pathway.

In conclusion, the results of the current study demonstrate that MAPK activity in cumulus cells, rather than the oocyte, is required for gonadotropin-induced meiotic resumption. In cumulus cells at least one signaling pathway, e.g. cAMP-PKA, can activate MAPK and induce GVB. Moreover, MAPK activity in the cumulus cell is also required for gonadotropin-induced cumulus expansion, and this MAPK-dependent step takes place downstream of an oocyte-secreted, cumulus expansion-enabling factor.


   Acknowledgments
 
We thank Dr. Martin M. Matzuk for kindly providing GDF-9, Drs. Wes G. Beamer, Robert Taft, Carrie Marin-Bivens, Janice Noveroske, and Maria M. Viveiros for their helpful discussions and critical reading of the manuscript. We are very grateful to Jennifer S. Smith of Biological Services for the assistance in preparing the photomicrographs. The PMSG and hFSH used in this study were generously provided by the National Hormone and Pituitary Program, NIDDK.


   Footnotes
 
This work was supported by NCI Grant CA-62392. The Scientific Services of The Jackson Laboratory receive support from a Cancer Core Grant (CA-34196) from the NCI.

Abbreviations: 8-Br-cAMP, 8-Bromo-5'-cAMP; CEI, cumulus expansion index; DO, denuded oocytes; EGF, epidermal growth factor; GDF-9, growth differentiation factor-9; GV, germinal vesicle; GVB, germinal vesicle breakdown; HA, hyaluronic acid; hFSH, human recombinant FSH; HX-medium, medium containing 4 mM hypoxanthine; MAPKK/MEK, MAPK kinase; OCC, oocyte-cumulus cell complexes; OOX, oocytectomized.

Received November 5, 2001.

Accepted for publication February 13, 2002.


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 Materials and Methods
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