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Endocrinology Vol. 146, No. 10 4437-4444
Copyright © 2005 by The Endocrine Society

Cyclic Adenosine 3',5'-Monophosphate-Dependent Activation of Mitogen-Activated Protein Kinase in Cumulus Cells Is Essential for Germinal Vesicle Breakdown of Porcine Cumulus-Enclosed Oocytes

Cheng-Guang Liang, Li-Jun Huo, Zhi-Sheng Zhong, Da-Yuan Chen, Heide Schatten and Qing-Yuan Sun

State Key Laboratory of Reproductive Biology, Institute of Zoology (C.-G.L., L.-J.H., D.-Y.C., Q.-Y.S.), and Graduate School (C.-G.L., L.-J.H.), Chinese Academy of Sciences, Beijing 100080, China; and Department of Veterinary Pathobiology (Z.-S.Z., H.S.), University of Missouri-Columbia, Columbia, Missouri 65211

Address all correspondence and requests for reprints to: Qing-Yuan Sun, Ph.D., State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China. E-mail: sunqy1{at}yahoo.com; or sunqy{at}ioz.ac.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
MAPK plays an important role during meiotic maturation in mammalian oocytes, whereas the necessity of MAPK during meiotic resumption in porcine oocytes is still controversial. Here, by applying the method of ultracentrifugation to move the opaque lipid droplets to the edge of the oocyte, therefore allowing clear visualization of porcine germinal vesicles, oocytes just before germinal vesicle breakdown (GVBD) and those that had just undergone GVBD were selected for the assay of MAPK activation. Our results showed that phosphorylation of MAPK in oocytes occurred after GVBD in all three different culture models: spontaneous maturation model, inhibition-induction maturation model, and normal maturation model. Moreover, we found that activation of MAPK in cumulus cells but not in oocytes was essential for GVBD in cumulus-enclosed oocytes. Then the cross-talk between cAMP and MAPK in cumulus cells was investigated by using cell-type-specific phosphodiesterase (PDE) isoenzyme inhibitors. Our results showed that PDE3 subtype existed in oocytes, whereas PDE4 subtype existed in cumulus cells. PDE3 inhibitor prevented meiotic resumption of oocytes, whereas PDE4 inhibitor enhanced the ability of FSH or forskolin to activate MAPK in cumulus cells. We propose that increased cAMP resulting from inhibition of PDE3 in oocytes blocks GVBD, whereas increased cAMP resulting from inhibition of PDE4 activates MAPK pathway in cumulus cells, which is essential for GVBD induction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
FULLY GROWN MAMMALIAN oocytes are arrested at the diplotene stage of first meiotic prophase, which is termed germinal vesicle (GV) stage. GV stage-arrested oocytes can resume meiosis spontaneously when they are released from the inhibitory environment of follicles (1). They can also mature in vitro under the stimulation of LH (2), FSH (3), or epidermal growth factor (EGF) (4) when the spontaneous maturation is prevented by meiotic inhibitors, such as hypoxanthine (HX), a natural meiotic inhibitory substance existing in follicular fluids (5). Alternatively, another maturation model, in which gonadotropin and growth factor but not cAMP (6) elevating agents are supplemented, is commonly used (7).

MAPK, also termed ERK, plays a very important role in the process of oocyte maturation (1, 8). The most widely studied MAPKs are 44- and 42-kDa MAPK isoforms (ERK1 and ERK2, respectively), which are rapidly activated in response to various growth factors and tumor promoters in cultured mammalian cells (9, 10, 11, 12, 13). In porcine oocytes, it has been shown by us and others that MAPK exists in an inactive form in the GV stage, and it is activated around the time of germinal vesicle breakdown (GVBD) (14, 15, 16). Because it is difficult to judge the nuclear status due to the accumulation of dark lipid droplets in the cytoplasm and because the cell cycle progresses asynchronously, it is difficult to tell the relationship between MAPK activation and meiosis resumption. MAPK is reportedly activated before (17, 18, 19), during (6, 20), or after (15) GVBD or maturation promoting factor activation in porcine oocytes. The necessity of MAPK involvement in oocyte GVBD is contradictory when different in vitro culture models are used (21, 22, 23, 24). Besides, developmental potential of the oocyte is related to the presence of cumulus oophorus (25). Our previous study showed that the MAPK kinase (MEK) inhibitor that specifically inhibits the MAPK pathway had different effects on meiotic maturation of cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs) (21).

There is abundant evidence showing that the second-messenger cAMP (adenosine cAMP) plays an important role in controlling meiotic resumption in rodent oocytes (26, 27, 28, 29, 30). But the role of cAMP in the control of meiosis is less understood in domestic animals. Several agents that experimentally elevate oocyte cAMP transiently inhibit the spontaneous meiotic maturation of bovine oocytes isolated from their follicles in vitro (31, 32, 33, 34, 35, 36, 37). In contrast, meiotic resumption is induced in follicle enclosed rodent oocytes after injection of cAMP analogs into the follicular antrum (38) or exposure of the follicles to dibutyryl-cAMP (dbcAMP) (39), phosphodiesterase (PDE) inhibitors (40), or forskolin (41). These apparently opposing actions of cAMP were attributed to the differential localization of distinct PDE subtypes in oocyte and cumulus cells (i.e. PDE3 subtype in oocyte and PDE4 subtype in cumulus cells) (42, 43).

More recent studies have shown that LH- or FSH-stimulated MAPK activation is mediated by cAMP-dependent protein kinase A in granulose cells (44, 45, 46). But the reagents used previously such as HX, dbcAMP, or forskolin that elevate cAMP levels have an effect on both cumulus cells and oocytes, which makes it difficult to investigate the relationship between cAMP and MAPK in cumulus cells when CEOs are cultured. Fortunately, the availability of subtype-specific PDE inhibitors (such as PDE3 inhibitor milrinone, cilostamide, and PDE4 inhibitor rolipram) provides a new opportunity for more extensive examination of oocyte maturation mechanisms and represents new and powerful experimental tools for investigating oocyte-follicular cell interactions.

The aim of the present study was to examine whether the activation of MAPK in porcine oocytes and cumulus cells participate in the process of GVBD in different culture models. By using PDE3- and PDE4-specific inhibitors, we also investigated the cross-talk between cAMP and MAPK in cumulus cells during oocyte meiosis resumption induction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Chemicals
All chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Stock solutions of U0126 (10 mmol/liter; Calbiochem, La Jolla, CA), forskolin (50 mmol/liter), milrinone (50 mmol/liter), cilostamide (50 mmol/liter), rolipram (50 mmol/liter), and dbcAMP (0.1 mol/liter) were prepared in dimethyl sulfoxide. Stock solutions of 8-bromoadenosine-cAMP (8-Br-cAMP) (0.1 mol/liter; Calbiochem) and equitorial (Rp)-8-Br-cAMP (0.1 mol/liter; Calbiochem) were prepared in 0.9% (wt/vol) NaCl. All stock solutions were frozen at –20 C in dark boxes. The chemicals were diluted and supplemented to the culture medium approximately 1 h before the oocytes were added in for culture.

Collection and preparation of porcine oocytes
Porcine ovaries were collected from gilts at a local slaughterhouse and transported to the laboratory within 1.5–2 h in a thermos bottle containing warm (30–35 C) saline [0.9% (wt/vol) NaCl supplemented with 40 IU/ml penicillin G and 50 µg/ml streptomycin sulfate]. The contents of follicles measuring 3–5 mm in diameter were aspirated with an 18-gauge needle fixed to a 20-ml disposable syringe and pooled in 50 ml conical tubes (Falcon, Franklin Lakes, NJ). After sedimentation, the sediment was washed with 20 mmol/liter HEPES-buffered tissue culture medium-199 (H-TCM-199; Life Technologies, Inc., Grand Island, NY) supplemented with penicillin G (100 IU/ml), streptomycin sulfate (100 µg/ml), BSA (fraction V; 4 mg/ml; Calbiochem), and HX (4 mmol/liter). H-TCM-199 was supplemented with the meiotic inhibitor HX to prevent premature commitment to meiotic progression during the collection of oocytes, and subsequent removal of inhibitors does not change the frequency of maturation and development (data not shown).

Oocytes with at least four layers of intact, compact cumulus cells were recovered under a stereomicroscope and transferred to 35 mm petri dishes (Falcon) containing culture medium for three washes before culture. Whereas DOs need to be cultured, CEOs were mechanically denuded using a vortex instrument to remove all cumulus cells surrounding the oocytes in 0.5 ml H-TCM-199 containing 300 IU/ml hyaluronidase.

Oocyte culture
Culture of CEOs and DOs was carried out in 4-well dishes (Nunc, Roskilde, Denmark) at 39 C in an atmosphere of 5% CO2 in air and saturated humidity. Three kinds of maturation culture models were used in our study: 1) spontaneous maturation model without any gonadotropin or growth factor; the oocytes were cultured in TCM-199 medium supplemented with 0.23 mmol/liter of sodium pyruvate, 2 mmol/liter glutamine, 3 mg/ml lyophilized crystallized BSA, 100 IU/ml penicillin G, and 50 µg/ml streptomycin sulfate; 2) inhibition-induction maturation model, which mimics intact follicle (4 mmol/liter HX and 100 IU/liter of FSH were supplemented in the culture medium that was used in the spontaneous maturation model); and 3) normal maturation model containing gonadotropins and growth factor; 0.57 mmol/liter cysteine, 0.5 µg/ml FSH, 0.5 µg/ml LH, and 10 ng/ml epidermal growth factor were supplemented to the culture medium that was used in the spontaneous maturation model.

Nuclear status examination
Nuclear status of porcine oocytes was examined with the orcein staining method, which was carried out as described previously (47). Briefly, denuded oocytes were mounted on slides, fixed in acetic acid/ethanol (1:3 vol/vol) for at least 24 h, stained with 1% orcein, and examined with a phase-contrast microscope (Nikon, Tokyo, Japan). Oocytes showing clear nuclear membranes (GV) were classified as GV stage oocytes and those that did not show nuclear membranes were classified as GVBD.

Oocyte ultracentrifugation
Because the nuclear status of the porcine oocytes used for Western blot analysis cannot be determined with the orcein staining method, we developed an ultracentrifugation method to select GV and GVBD oocytes for Western blot analysis. Denuded oocytes were transferred to a 0.5-ml tube (Eppendorf, Hamburg, Germany) containing 0.3 ml TCM-199 medium supplemented with 4 mmol/liter HX and then centrifuged at 10,000 rpm for 5 min. Porcine GVs can be visualized when lipid droplets are centrifuged to the edge of the oocyte. The oocytes were examined with a phase-contrast microscope equipped with a micromanipulator (Narishige, Tokyo, Japan). In the process of examination, GVs could be visualized after turning the oocytes with the micromanipulator. Oocytes that had a visible GV were collected and classified as GV group and the others without visible GV were classified as GVBD group (see Fig. 1Go).



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  		FIG. 1. GV of porcine oocyte is visible after ultracentrifugation. Porcine oocytes were centrifuged at 10,000 rpm for 5 min to visualize the GV status. GV could be visualized with a phase-contrast microscope after ultracentrifugation. At times the GV is obscured by the dark lipid droplets on one side of the oocyte, but after rolling the oocyte with the micromanipulator, the GV will become visible. A, Oocyte with a germinal vesicle (arrow); a) amplified germinal vesicle (arrow). B, Oocytes that underwent GVBD had no visible GV after rolling with micromanipulator.

 
Electrophoresis and Western blot analysis
Oocytes for Western blot analysis were denuded by vortexing in 300 IU/ml hyaluronidase to remove the cumulus cells. Cumulus cells for Western blot analysis were collected by removing the oocytes from CEOs by mechanical repeated pipetting with small-bore Pasteur pipettes. MAPK activity in 50 oocytes or cumulus cells derived from 20 CEOs was determined indirectly by detection of phosphorylated (active) forms of MAPK using Western blot analysis as described by Sun and Fan (48). Phosphorylated MAPK was detected by using a polyclonal mouse antihuman pERK1/2 antibody. The secondary antibody was horseradish peroxidase (HRP)-conjugated goat antimouse IgG. The total amount of ERK2 on the same membrane was assayed by using a polyclonal rabbit anti-ERK2 antibody after stripping off the initial bound antibodies. In this reaction, HRP-conjugated goat antirabbit IgG was used as the secondary antibody. For PDE3A and PDE4A1 detection, total protein of 500 GV oocytes or cumulus cells derived from 500 CEOs were separated by SDS-PAGE with an 8% separating gel. Polyclonal goat antihuman PDE3A or PDE4A1 antibody and HRP-conjugated rabbit antigoat IgG were used as the primary and secondary antibodies, respectively. All antibodies used in this study were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The gel bands were analyzed with ImageQuant TL software (Amersham Biosciences, Piscataway, NJ).

Statistical analysis
All percentages from three repeated experiments are expressed as mean ± SEM. All frequencies were subjected to arcsine transformation. The transformed data were statistically compared by ANOVA using SPSS 10.0 software (SPSS Inc., Chicago, IL) followed by the Student-Newman-Keuls test. Differences of P < 0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Activation of MAPK in porcine oocytes does not play a role in meiosis resumption, regardless of the culture models employed
Because the progression of cell cycle in porcine oocytes is asynchronous, it is necessary to ascertain the exact time of GVBD in different culture models. When the GVBD rate was more than 5%, we regarded this time as GVBD begin-to-occur time point. As shown in Fig. 2Go, our results showed that in the spontaneous maturation model, GVBD of CEOs began at 36 h and ended at 42 h, whereas GVBD of DOs began at 16 h and ended at 24 h. In the inhibition-induction maturation model, GVBD of CEOs began at 22 h and ended at 32 h, whereas GVBD of DOs began at 22 h and ended at 30 h. In the normal maturation culture model, the occurrence of GVBD of CEOs and DOs began at 16 h and ended at 24 h.



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  		FIG. 2. GVBD of porcine oocytes cultured in different maturation models. GVBD of porcine oocytes in different maturation models were assessed with the orcein-staining method. Approximately 50 CEOs or DOs in each group were cultured in three different maturation models and collected every 2 h for fixation and orcein staining. The time table of porcine oocyte GVBD was determined to figure out the duration in which most of the oocytes undergo GVBD in different culture models. Oocytes of GV stage or GVBD stage collected during this time were regarded as being in the process of imminent GVBD or just having undergone GVBD, respectively. Data are presented as the percentage of GVBD (mean ± SEM of three independent experiments).

 
To determine the relationship between MAPK activation and GVBD in oocyte, ultracentrifugation was used to select the oocytes in the process of imminent GVBD and those that had just undergone GVBD based on the GVBD timetable. Then the two categories of oocytes collected for each time point were used for MAPK analysis. As shown in Fig. 3Go, our results showed that whatever the culture models used, no active MAPK was detected in the oocytes that would undergo GVBD imminently, but once GVBD occurred (oocytes without visible GV), the phosphorylation of MAPK was detected in these oocytes. These results showed that activation of MAPK occurred after GVBD in porcine oocytes, regardless of which kind of culture model was used. Phosphorylation of MAPK in five GVBD oocytes could be detected in control groups (Fig. 3Go, normal maturation model), indicating that the Western blot system was sensitive enough for active MAPK detection.



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  		FIG. 3. Phosphorylation of MAPK in porcine oocytes occurs after GVBD. Oocytes cultured in spontaneous maturation model, inhibition-induction maturation model, and normal maturation model were divided into two groups with respect to the presence of GV after ultracentrifugation. Protein extract of 50 oocytes was subjected to Western blot analysis with anti-p-ERK1/2 and anti-total-ERK2 antibodies. Protein extract from five GVBD oocytes in normal maturation model was adopted as control to test the sensitivity of the Western blot system. The results of one representative of three independent experiments are presented. Regardless of the culture models used, active MAPK could be detected in GVBD oocytes but not GV oocytes. This indicates that activation of MAPK in oocyte does not participate in GVBD.

 
Phosphorylation of MAPK in cumulus cells is inhibited by U0126
Our previous study showed that the MAPK pathway’s specific inhibitor U0126 could inhibit GVBD of CEOs but not DOs (21). Thus, activation of MAPK in oocytes may not participate in GVBD. We assume that active MAPK in cumulus cells but not in oocytes is essential for GVBD. So the effect of U0126 on phosphorylation of MAPK in cumulus cells of CEOs was investigated. As shown in Fig. 4Go, in the group without U0126, activation of MAPK in cumulus cells could be observed immediately 1 h after culture, and an increase in MAPK activity was observed when the culture time was prolonged, whereas active MAPK in cumulus cells could not be detected up to 4 h of culture in the U0126-supplemented group. Our next goal was to investigate the pathway of MAPK activation in cumulus cells.



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  		FIG. 4. U0126 inhibits the phosphorylation of MAPK in cumulus cells derived from CEOs. CEOs were cultured in the inhibition-induction maturation model medium supplemented with or without 10 µmol/liter U0126. At each time point, cumulus cells derived from 20 CEOs were used for Western blot analysis with anti-p-ERK1/2 and anti-total-ERK2 antibodies. The results of one representative of three independent experiments are presented. In the control group without U0126, MAPK in cumulus cells could be totally activated at 4 h. But activation of MAPK did not occur up to 4 h in the U0126 group.

 
PDE3 exists in oocytes, whereas PDE4 exists in cumulus cells
Cellular cAMP could be elevated through the inhibition of PDE. To study the cross-talk between cAMP and MAPK in porcine cumulus cells, we investigated the expression of two PDE isoforms, PDE3 and PDE4, in porcine oocytes and cumulus cells by Western blot analysis. As shown in Fig. 5Go, PDE3A could be detected in oocytes but not in cumulus cells, whereas PDE4A1 could be detected in cumulus cells but not in oocytes.



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  		FIG. 5. Expression of PDE3 and PDE4 in oocytes and cumulus cells. The expression of PDE3A and PDE4A1 in 500 oocytes and cumulus cells derived from 500 CEOs was detected with Western blot analysis. The results of one representative of three independent experiments are presented. PDE3A could be detected in oocytes but not in cumulus cells, whereas PDE4A1 could be detected in cumulus cells but not oocytes.

 
GVBD of porcine CEOs is inhibited by PDE3 inhibitor milrinone and cilostamide but not by PDE4 inhibitor rolipram
We further assessed the effects of PDE3 inhibitors milrinone and cilostamide as well as PDE4 inhibitor rolipram on GVBD of porcine CEOs. As shown in Fig. 6Go, low concentrations (1 µmol/liter) of PDE inhibitors did not affect GVBD. But when CEOs were incubated in the medium supplemented with 10 µmol/liter PDE3 inhibitor milrinone or cilostamide, the percentage of oocytes undergoing GVBD is apparently decreased (51.7 and 52.4%, respectively). When higher concentrations (50 µmol/liter) of PDE inhibitors were used, the group treated with milrinone had a lower rate of GVBD (24.8%), whereas treatment with PDE4 inhibitor rolipram did not affect GVBD, even at 50 µmol/liter. Is the inhibitory effect of PDE3 inhibitors on GVBD due to their toxicity to the oocyte? The reversibility of GVBD was assessed by culturing CEOs for an additional 24 h in the medium without any PDE inhibitors. The results showed that the meiotic arrest was fully reversible after PDE inhibitors were removed from the culture medium (Fig. 6Go).



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  		FIG. 6. Effects of PDE3 and PDE4 inhibitors on GVBD of porcine CEOs. The effects of different concentrations of PDE3 inhibitor milrinone (Mil), cilostamide (Cil), and PDE4 inhibitor rolipram (Rol) on GVBD of CEOs were evaluated. Porcine CEOs were cultured for 24 h in spontaneous maturation medium supplemented with 100 IU FSH/liter and 1, 10, or 50 µmol/liter PDE inhibitors. The reversibility of the PDE inhibitors on the CEOs was assessed by an additional culture for 24 h without any PDE inhibitors in the culture medium. The group without any PDE inhibitor was adopted as control. GVBD was observed at 24 or 48 h by using the orcein staining method. Approximately 50 oocytes were included in each group. Data are presented as the percentage of GVBD (mean ± SEM of five independent experiments). Where there are no common letters over the bars, the groups were significantly different (P < 0.05). High concentration (10 or 50 µmol/liter) of PDE3 inhibitor but not PDE4 inhibitor could arrest GVBD. The inhibition effect could be reversed after removing the inhibitor.

 
PDE4 inhibitor but not PDE3 inhibitors enhances the ability of FSH-induced activation of MAPK in cumulus cells during the initial culture period
With the above hypothesis in mind, we wanted to know whether PDE3 inhibitor and PDE4 inhibitor have different effects on FSH-induced phosphorylation of MAPK in cumulus cells. As shown in Fig. 7Go, in the control group, the activity of MAPK had been increasing and reached the maximum at 6 h. MAPK was activated rapidly in the group supplemented with rolipram for 1 h of culture. MAPK in the groups supplemented with PDE3 inhibitor was not fully activated even when cultured for 6 h. We also found that a decreased activity of MAPK was present at 6 h in the rolipram group.



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  		FIG. 7. Effect of PDE3 inhibitors and PDE4 inhibitor on FSH-induced phosphorylation of MAPK in porcine cumulus cells. Porcine CEOs were cultured in 100 IU/liter FSH-supplemented spontaneous maturation medium containing 50 µmol/liter PDE3 inhibitor milrinone (Mil) or cilostamide (Cil), or PDE4 inhibitor rolipram (Rol). After culture for 1, 3, or 6 h, cumulus cells derived from 20 CEOs were used for Western blot analysis with anti-p-ERK1/2 and anti-total-ERK2 antibodies. The group without inhibitors was adopted as control. The results of one representative of three independent experiments are presented. Data are presented as the numerical densitometry values (mean ± SEM of three independent experiments). PDE4 inhibitor rolipram could enhance FSH-induced activation of MAPK in the initial culture period, whereas PDE3 inhibitors had no such effect.

 
The ability of FSH or forskolin to activate MAPK in cumulus cells of porcine CEOs is enhanced by PDE4 inhibitor rolipram
Because PDE4 locates in cumulus cells and PDE4 inhibitor participates in FSH-induced activation of MAPK in cumulus cells, another question was raised. Does cAMP pathway participate in the process of rolipram-stimulated MAPK in cumulus cells? The effects of different cAMP elevators and inhibitors on the activation of MAPK in cumulus cells of porcine CEOs were investigated. As shown in Fig. 8Go, in the group without cAMP elevators, neither milrinone nor rolipram by itself could activate MAPK, whereas in the group supplemented with forskolin, phosphorylation of MAPK could be observed, and this effect could be enhanced by rolipram but not milrinone. Furthermore, we found that rolipram could enhance the ability of FSH to activate MAPK, whereas milrinone has no such effect. Other cAMP elevators such as dbcAMP, 8-Br-cAMP, and cAMP inhibitor such as Rp-8-Br-cAMP, which have no cell-type specificity, were also tested. The results showed that MAPK could be activated by dbcAMP and 8-Br-cAMP, whereas FSH-stimulated MAPK could be inactivated by Rp-8-Br-cAMP. These results indicate that cAMP participates in rolipram-induced MAPK activation in porcine cumulus cells.



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  		FIG. 8. Effect of cAMP modulators on the activation of MAPK in cumulus cells of porcine CEOs. The effects of cAMP activators [FSH, forskolin (For), milrinone (Mil), cilostamide, rolipram (Rol), dbcAMP, and 8-Br-cAMP] and inhibitor (Rp-8-Br-cAMP) on the phosphorylation of MAPK in cumulus cells were assessed. CEOs were cultured in spontaneous maturation medium that was supplemented with the following chemicals: 1) control group without any activators or inhibitors; 2) 50 µmol/liter milrinone; 3) 50 µmol/liter rolipram; 4) 50 µmol/liter forskolin; 5) 50 µmol/liter forskolin plus 50 µmol/liter milrinone; 6) 50 µmol/liter forskolin plus 50 µmol/liter rolipram; 7) 100 IU/liter FSH plus 50 µmol/liter milrinone; 8) 100 IU/liter FSH plus 50 µmol/liter rolipram; 9) 1 mmol/liter dbcAMP; 10) 0.1 mmol/liter 8-Br-cAMP; and 11) 100 IU/liter FSH plus 0.1 mmol/liter Rp-8-Br-cAMP. Cumulus cells derived from 20 CEOs cultured for 1 h were used for the detection of p-ERK1/2 and total-ERK2. The results of one representative of four independent experiments are presented. Data are presented as the numerical densitometry values (mean ± SEM of four independent experiments). PDE4 inhibitor by itself could not activate MAPK. cAMP activators could induce phosphorylation of MAPK in cumulus cells. PDE4 inhibitor could enhance the ability of forskolin or FSH to activate MAPK, whereas PDE3 inhibitors had no such effect. Furthermore, cAMP inhibitor could reduce the activity of MAPK induced by FSH.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
In this study, we used three culture models and used ultracentrifugation of porcine oocytes and the specific MEK inhibitor U0126 to provide direct evidence showing that active MAPK in cumulus cells but not in oocytes participates in GVBD of CEOs. The use of cell type-specific PDE inhibitors and other cAMP modulators shows that elevation in cAMP is related to activation of MAPK in cumulus cells.

Ultracentrifugation of oocytes is used to determine cell cycle status before biochemical analysis
A technical problem should be overcome when analyzing the correlation between MAPK activation and oocyte GVBD, i.e. we need to know the development status of porcine oocytes before performing the MAPK assay. In oocytes from the pig, cow, goat, and horse, the cytoplasm is opaque and the nucleus is invisible in living oocytes. Due to the long duration of the GV stage, GVBD in these oocytes is somewhat asynchronous. In previous Western blot analysis, proteins from the oocytes of GV stage and GVBD stage coexisted in the same sample. For this reason, we cannot discern which kind of oocytes (GV oocytes or GVBD oocytes or both of them) contains the phosphorylated MAPK. In this study, the method of ultracentrifugation was used to tell exactly the stage of porcine oocytes before protein extraction. This method made it possible to determine the activation of MAPK before or after the occurrence of GVBD, and it also allows us to determine whether active MAPK activation in oocytes is essential for GVBD.

The activation of MAPK in cumulus cells but not in oocytes is essential for GVBD as revealed by culture of CEOs and DOs in different culture systems
Whether MAPK participates in GVBD of porcine oocytes has been debated for a long time. Our previous studies showed that artificial activation of MAPK by okadaic acid could significantly accelerate GVBD of porcine oocytes (49). However, injection of antisense RNA of porcine c-mos protein, an upstream kinase of MAPK, into porcine oocytes does not affect GVBD in spontaneous meiotic resumption in denuded oocytes, although MAPK phosphorylation is completely inhibited (23). In the present study, we found that MAPK in oocytes was activated after GVBD in both CEOs and DOs in three different systems, but it was activated before GVBD in cumulus cells during in vitro culture, and its activation as well as GVBD of CEOs was blocked by U0126. Similar results were obtained by using MostmlEv/MostmlEv (Mos-null) mice, in which MAPK activation occurs only in the cumulus cells but not in the oocyte, whereas GVBD occurs normally (24). MAPK is activated indirectly by MOS in oocytes (50, 51). But in cumulus cells, MAPK is activated indirectly by RAS/RAF (another MAPK kinase kinase) signaling pathway. Because the activation of MAPK is directly mediated by MEK, which could be inhibited by U0126 in both oocytes and cumulus cells (52), it is highly possible that MAPK activation in oocytes is not required for meiotic resumption, but activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of porcine and mouse CEOs.

Different isoforms of PDE exist in porcine oocytes and cumulus cells
The second-messenger cAMP plays a crucial role in the maturation of mammalian oocytes. cAMP can be elevated by non-cell-specific reagents such as forskolin (32), dbcAMP (32, 33, 37), IBMX, and HX (53) in both oocyte and cumulus cells when CEOs are cultured. It was recently reported that actions of PDE4 inhibitors in follicle cells enhance effects of LH on oocyte maturation, whereas PDE3 inhibitors act directly in oocytes, increasing cAMP level and suppressing meiotic progression (42, 54). It was also reported that treatment with PDE4 inhibitor rolipram resulted in increase in the level of cAMP in granulosa cells, whereas treatment with the PDE3 inhibitor milrinone resulted in increase in the level of cAMP in oocytes (42, 43, 54, 55). In our study, what has been observed in murine and bovine oocytes also occurs in porcine oocytes. These results let us understand the different effects of FSH on cAMP levels in the two follicular compartments and the mechanisms of the resumption of meiosis.

Cross-talk between cAMP and MAPK in cumulus cells is required for inducing oocyte meiosis resumption
Various studies suggest that LH promotes an increase in cAMP levels within the granulosa cell compartment and a decrease in cAMP in the oocyte, thus inducing the resumption of meiosis as well as cumulus expansion (42, 56, 57, 58). It has also been reported that MAPK is activated in ovarian granulosa cells in response to GnRH (59, 60), LH (44), and FSH (61). Thus, we investigated the relationship between increase in cAMP and activation of MAPK in cumulus cells. FSH and forskolin-induced activation of MAPK could be enhanced by cumulus-specific PDE4 inhibitor rolipram but not oocyte-specific PDE3 inhibitor milrinone or cilostamide. We assumed that activation of MAPK in cumulus cells depended on the elevation of cAMP stimulated by FSH or forskolin. Rolipram inhibited the PDE4-mediated degradation of cAMP and thus activated MAPK, whereas Rp-8-Br-cAMP could inhibit cAMP and thus inhibited activation of MAPK. Besides, the activation of MAPK could be induced by non-cell-specific cAMP activators dbcAMP and 8-Br-cAMP. Although the extensive network of gap junctions between the oocyte and the cumulus cells could facilitate the transfer of cAMP between these two compartments (62), a high level of cAMP induced by non-cell-specific or cell-specific cAMP activators in the cumulus cells may induce the release of a signal or signals that trigger meiotic resumption despite the presence of a high level of cAMP in the oocyte.

We also noted that rolipram by itself did not have the ability to activate MAPK if FSH or forskolin was not supplemented (Fig. 8Go). The elevation of cAMP caused by rolipram is due to its ability to inhibit the activity of PED4. The density of active MAPK in the group supplemented with FSH and rolipram is much higher than that in the group supplemented with forskolin and rolipram. Our previous study has shown that MAPK in mouse cumulus cells could be activated by FSH through the protein kinase C pathway (3). Thus, MAPK could be activated by FSH through protein kinase A and protein kinase C as well as other unidentified pathways in porcine cumulus cells.

Future questions
The downstream targets of MAPK in cumulus cells are now under investigation by several laboratories. Recent studies found that MAPK mediates LH-induced oocyte maturation by interrupting cell-to-cell communication through phosphorylation of connexin43 within the ovarian follicle (63). It was also found that cumulus expansion was related to the activation of MAPK (64). Others noticed that the G protein-coupled receptor 3 is a link in communication between the somatic cells and oocytes of the ovarian follicle and is crucial for the regulation of meiosis (65). The relationship between MAPK and G protein-coupled receptor 3 receptor is another interesting aspect. Recently it has been suggested that LH induces the activation of the ovarian granulosa EGF receptors through enhanced expression of certain EGF family members (66). Whether activation of MAPK is induced by EGF family members needs further investigation (67). In our study, the activity of MAPK was decreased in the cumulus cells supplemented with FSH and PDE4 inhibitor rolipram at 6 h, and further studies are needed to explain this phenomenon.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
In this study we have provided direct evidence showing that activation of MAPK in cumulus cells but not in oocytes is essential for GVBD of porcine CEOs. Increased cAMP resulting from inhibition of PDE3 in oocyte blocks GVBD, whereas increased cAMP resulting from inhibition of PDE4 in cumulus cells induces GVBD through MAPK pathway (Fig. 9Go).



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  		FIG. 9. A proposed model for GVBD induction of porcine CEOs. FSH-dependent elevation of cAMP can be enhanced by PDE4 inhibitor in cumulus cells. The elevated cAMP could activate MAPK, which in turn induces the release of a signal or signals that trigger meiotic resumption. PDE3 inhibitor participates in elevation of cAMP in oocytes and blocks meiotic resumption of porcine CEOs.

 


   Acknowledgments
 
We thank Qiang Wang, Shen Yin, Jun-Shu Ai, Zhen-Bo Wang, Chang-Long Nan, Zhen-Jun Zhao, and Gang Zhang for their expert technical assistance as well as Yi Hou and Xiang-Fen Song for their preparation of experimental materials.


   Footnotes
 
This work was supported by the National Natural Science Foundation of China (30225010,30430530) and the Special Funds for Major State Basic Research Project (973) of China (G1999055902).

First Published Online July 7, 2005

Abbreviations: 8-Br-cAMP, 8-Bromoadenosine-cAMP; CEO, cumulus-enclosed oocyte; dbcAMP, dibutyryl-cAMP; DO, denuded oocyte; EGF, epidermal growth factor; GV, germinal vesicle; GVBD, germinal vesicle breakdown; HRP, horseradish peroxidase; H-TCM-199, HEPES-buffered tissue culture medium-199; HX, hypoxanthine; MEK, MAPK kinase; PDE, phosphodiesterase.

Received March 16, 2005.

Accepted for publication June 27, 2005.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
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