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Endocrinology Vol. 142, No. 7 3187-3197
Copyright © 2001 by The Endocrine Society

ARTICLES

Regulation of Prostaglandin-Endoperoxide Synthase 2 Messenger Ribonucleic Acid Expression in Mouse Granulosa Cells during Ovulation1

Ieuan M. Joyce2, Frank L. Pendola, Marilyn 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
 
Normal ovulation in mice requires PG-endoperoxide synthase 2 (cyclooxygenase-2; COX-2) expression. This study examined the role of the oocyte and other factors in regulating steady state levels of COX-2 messenger RNA (mRNA) in granulosa cells. Multiphasic changes in the expression pattern of COX-2 mRNA were found, with peaks of expression 4 and 12 h after hCG treatment. Changes in relative expression levels in cumulus cells and mural granulosa cells occurred over time, with similar mRNA levels at 4 h, but higher levels in cumulus cells compared with mural granulosa cells at 8 and 12 h post-hCG. In cultured mural granulosa cells, LH, FSH, and oocytes promoted COX-2 mRNA expression concurrent with the first expression peak in vivo. At the same time, FSH, but not LH, treatment of cultured cumulus-oocyte complexes (COC) promoted COX-2 mRNA expression in cumulus cells. This response of cumulus cells to FSH treatment was largely dependent on the presence of either fully grown germinal vesicle stage or maturing oocytes, but not growing oocytes. At 8 h, COX-2 mRNA expression in FSH-stimulated COC was lower than at 4 h; however, oocyte coculture promoted COX-2 mRNA expression in cumulus cells. No second peak in expression occurred in cultured COC. However, coculture of COC with follicle walls promoted COX-2 mRNA expression in cumulus cells 12 h post-hCG; an effect augmented by oocytes. Therefore, the oocyte resident within ovulatory follicles produces a factor(s) that promotes expression of COX-2 mRNA by cumulus cells and possibly by mural granulosa cells. Thus, the oocyte probably plays an important role in promoting ovulation. However, the multiphasic changes in the pattern of COX-2 expression appear orchestrated by non-oocyte-derived factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PREOVULATORY surge of LH initiates complex multiphasic changes in gene expression patterns in preovulatory follicles. In the mouse these changes culminate in ovulation and formation of the corpus luteum within 12–14 h. One gene that shows a multiphasic expression pattern in granulosa cells in many species after the LH surge is Ptgs2, or PG-endoperoxide synthase 2, commonly known as cyclooxygenase-2 (COX-2) (1, 2, 3). COX-2, along with PGendoperoxide synthase 1 (COX-1), a separate gene product, catalyze the rate-limiting step in the conversion of arachidonate to prostanoids (4). A role for COX-2 in the ovulatory process has been demonstrated. For example, NS398, a specific inhibitor of COX-2, suppresses the ovulation rate in rats (5). Furthermore, Ptgs2tm1Jedl mice, which do not express COX-2, have defects in cumulus expansion and stigmata formation and display severely reduced ovulatory capacity (6). On the other hand, Ptgs1tm1Unc mice, which do not express COX-1, show no apparent abnormal ovarian phenotype (7). It is therefore likely that the production of prostanoids after the expression of COX-2 is an important step in the ovulatory process.

Granulosa cells in preovulatory and ovulatory follicles can be divided into two major subpopulations: cumulus cells and mural granulosa cells. Cumulus cells surround and are intimately associated with the oocyte, whereas mural granulosa cells line the follicle wall. In ovulatory follicles after the LH surge, cumulus cells undergo a process of separation and mucification (termed expansion) before expulsion from the follicle at the time of ovulation. On the other hand, mural granulosa cells luteinize after the LH surge, and eventually comprise part of the corpus luteum. Not surprisingly, the two granulosa cell subpopulations show distinct gene expression patterns. For example, Kitl (KIT ligand) messenger RNA (mRNA), Lhcgr (LH/CG receptor) mRNA, and Plau (urokinase plasminogen activator) are all expressed more highly mural granulosa cells than in cumulus cells (8, 9, 10). On the other hand, Has2 (hyaluronan synthase 2) and Ptgerep2 (PGE receptor EP2) mRNA are expressed more highly in cumulus cells (3, 11). Evidence from in situ hybridization studies suggests that COX-2 mRNA expression may also be higher in cumulus cells than in mural granulosa cells in mice (3, 12).

For a number of genes that are expressed at different levels in cumulus cells and mural granulosa cells, there is evidence that the oocyte may play a role in promoting the level of expression found in the cumulus cell phenotype. For example, fully grown oocytes suppress LH receptor and KL mRNA expression and promote hyaluronic acid synthesis in cumulus cells in vitro (13, 14, 15). One factor that may mediate these actions of oocytes is growth differentiation factor 9 (GDF-9). The gene for this factor is expressed specifically in oocytes in the ovary and is essential for normal follicle development beyond the primary stage (16, 17). Studies using recombinant GDF-9 have shown that the action of this protein in vitro is in many respects similar to that of fully grown oocytes (18, 19, 20). Interestingly, recombinant GDF-9 also up-regulates COX-2 mRNA expression in cultured mural granulosa cells (18, 21), suggesting that the oocyte may play a role in promoting the expression of COX-2. The evidence that COX-2 mRNA expression in granulosa cells is an important event during ovulation raises the intriguing possibility that the oocyte may play a pivotal role in promoting the ovulatory process.

This study was therefore undertaken to examine the likely role of the oocyte in the regulation of COX-2 mRNA expression in granulosa cells of ovulatory follicles. In initial studies using tissue collected 4 h after an ovulatory dose of hCG, we failed to confirm the observation that COX-2 mRNA expression is higher in cumulus than in mural granulosa cells. For this reason and because of the multiphasic nature of the ovulatory process, particular attention was paid to examining the kinetics of COX-2 mRNA expression in the two granulosa cell subpopulations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Granulosa cell and oocyte isolation and culture
A model system employing exogenous gonadotropin-stimulated prepubertal (C57BL/6J x SJL/J)F1 mice was used to study follicle development during the periovulatory period. This system enabled examination of ovarian tissues at defined stages of the ovulatory process. Briefly, 20-day-old mice were treated with 5 IU PMSG (provided by the NIDDK) to stimulate preovulatory follicular development. After 44–48 h, mice received an injection of 5 IU hCG (Sigma, St. Louis, MO). At this point after PMSG administration, hCG treatment has the effect of mimicking an endogenous LH surge, stimulating preovulatory follicles to undergo ovulatory follicle development and ultimately ovulation 12–16 h later. Depending on the experiment, tissue was collected 44–48 h after PMSG treatment (i.e. corresponding to the time of hCG treatment), and 2, 4, 8, 12, and 16 h after hCG treatment.

All cultures used medium 199 medium plus 3 mg/ml BSA (Life Technologies, Inc., Gaithersburg, MD) maintained at 37 C in a humidified 5% carbon dioxide, 5% oxygen, and 90% nitrogen atmosphere as previously described for the culture of COC and mural granulosa cells (14). The majority of cultures used 96-well tissue culture plates with a medium volume of 60 µl. Follicle shells were cultured under washed mineral oil in 35-mm petri dishes in an 80-µl volume of medium. Cultures were treated with recombinant human FSH (0.1–1.0 IU/ml; provided by Organon, Oss, The Netherlands), highly purified human LH (10–500 ng/ml; provided by Dr. A. Parlow, NIDDK), PGE2 (100 ng/ml; Sigma), mouse epidermal growth factor (EGF; 10 ng/ml; Collaborative Biomedical Products, Bedford, MA), recombinant human interleukin-1ß (IL-1ß; 0.01–1 ng/ml; Collaborative Biomedical Products, Bedford, MA), recombinant mouse IL-6 (5 ng/ml; Genzyme, Cambridge, MA), recombinant human tumor necrosis factor-{alpha} (5 ng/ml; Upstate Biotechnology, Inc., Lake Placid, NY); recombinant mouse interleukin-1 receptor antagonist (IL-1Ra; 200 ng/ml; provided by R & D Systems, Inc., Minneapolis, MN), and recombinant human insulin-like growth factor I (100 ng/ml; Upstate Biotechnology, Inc., Lake Placid, NY) as described in Results.

Cumulus-oocyte complexes (COC) and mural granulosa cell clumps were isolated 44–48 h after PMSG treatment or 2–12 h after hCG treatment by needling the ovaries in a 35-mm petri dish containing 3 ml culture medium. Ovulated COC collected 16 h after hCG treatment were isolated from the oviduct. After needling the ovaries, COC and mural granulosa cell clumps were picked up separately and washed through three changes of medium. Depending on the experiment, COC were either cultured intact, or clumps of cumulus cells were cultured after isolation from oocytes using a pipette-based oocytectomy procedure. This procedure was undertaken exactly as described previously for oocytectomizing preantral granulosa-oocyte complexes (14). Although some three-dimensional integrity of cumulus cell clumps is lost using the pipette-based oocytectomy procedure compared with microsurgical procedures (22), there is a considerable time saving. This efficiency was essential in the current experiments because of the large number of COC oocytectomized.

To isolate follicle shells, follicles were dissected from ovaries using 25-gauge needles, taking care to strip as much extraneous tissue as possible from the follicle. Follicles were then opened by slicing through a section of the follicle wall using dissecting needles. This procedure liberated the follicular fluid and the COC, and externalized parts of the inner follicle wall while maintaining the majority of the granulosa and thecal cells in a unified structure. This structure was then gently washed through three dishes to remove follicular fluid while retaining the follicle wall as a unit. Follicles treated in this way are subsequently referred to as follicle shells.

Oocytes at various developmental stages were used. Partly grown, meiotically incompetent oocytes, approximately 56 µm in diameter, were obtained from the preantral follicles of 12-day-old mice as described previously (13). Fully grown, meiotically competent oocytes, approximately 76 µm in diameter, were obtained by gentle pipetting of COC from PMSG-treated mice. All medium used for oocyte isolation contained 10 µM milrinone (Sigma), an inhibitor of the oocyte-specific phosphodiesterase-3 (23), to maintain oocytes at the germinal vesicle (GV) stage. Initial studies found no detectable effect of 10 µM milrinone on COX-2 mRNA expression (data not shown). After isolation, oocytes designated to be cultured while undergoing meiotic maturation were removed from milrinone-containing medium and washed three times. Depending on the experiment, release from phosphodiesterase-3 inhibition was timed to coincide with the start of the culture experiment or with hCG treatment of mice later killed for collection of cumulus cells.

Oocyte coculture experiments used fully grown (GV stage or maturing) oocytes at a concentration of 1.5 oocytes/µl culture medium and partly grown oocytes at a concentration of 3 oocytes/µl culture medium. These concentrations of fully and partly grown oocytes were chosen to be comparable on the basis of oocyte volume (24). Intact and oocytectomized COC were cultured at a concentration of 1.5 complexes/µl. Follicle shells were cultured at a concentration of 0.2 shells/µl. Culture time was between 4–14 h as described in Results.

In situ hybridization
In situ hybridization procedures were modified from those described by Manova et al. (25). Ovaries were fixed in 4% paraformaldehyde overnight, then washed, dehydrated, and embedded in paraffin wax. Sections 4 µm thick were mounted on SuperFrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA) and dried before being hydrated, postfixed in 4% paraformaldehyde, and treated with proteinase K and acetic anhydride. Slides were then subjected to a 3-h prehybridization at 65 C while RNA probe was prepared. The COX-2 RNA probe was generated from a mouse COX-2 complementary DNA provided by Dr. S. K. Dey. Antisense and sense RNA probes incorporating [{alpha}-33P]CTP (NEN Life Science Products, Boston, MA) were made with SP6 and T7 RNA polymerases, respectively, using MAXIscript kits (Ambion, Inc., Austin, TX). After probe preparation, slides were hybridized overnight at 65 C and washed after a 30-min ribonuclease (RNase) treatment at 37 C (1:40 dilution of RNase cocktail; Ambion, Inc.). Washing steps included immersion in 50% formamide/2 x SSC (standard saline citrate) at 65 C for 20 min and immersion in 0.1 x SSC at room temperature for 1 h. After washing, slides were dipped in NTB2 emulsion (Kodak, New Haven, CT) and exposed for 3–4 days before being developed and stained with hematoxylin and eosin. Hybridization signals using the sense COX-2 probe were at background levels across all sections tested. COX-2 mRNA expression was examined by in situ hybridization in ovaries from at least four mice at each time point.

RNase protection assay
RNase protection assay procedures were similar to those described previously (13, 14). Antisense COX-2 RNA probe was generated in a fashion similar to the RNA probe used for in situ hybridization, except incorporating [{alpha}-32P]CTP (NEN Life Science Products). [{alpha}-32P]CTPlabeled antisense RPL-19 RNA probe was also transcribed and included in all assays to allow differences in the quantity of mRNA between samples to be ameliorated mathematically (13, 26). Band intensity after electrophoresis was quantified using a phosphor imaging system (Fuji Photo Film Co., Ltd., Stamford, CT). Dose-response studies indicated that in a serially diluted sample, COX-2 mRNA was linearly related to total mRNA at least across the range of 20–400 ng mRNA/sample.

Statistical analysis
Experiments were repeated independently three to five times. The effect of treatment on COX-2 mRNA levels was assessed by ANOVA. Differences in background density between assays, as quantified by the phosphor imaging system, were large, thereby generating a high degree of interassay variation in average phosphor imaging units. Therefore, the data for individual replicates were first normalized so that the mean COX-2 mRNA levels for each replicate was equal to one. When a significant F ratio was defined by ANOVA, groups were compared using Fisher’s protected least significant difference post-hoc test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the kinetics of COX-2 mRNA expression in ovulatory follicles
In an initial survey using an RNase protection assay, steady state COX-2 mRNA levels were measured in cumulus and mural granulosa cells isolated 4 h after hCG treatment of PMSG-primed mice. At this time no difference between expression levels in the two cell subpopulations could be detected. Previous reports suggested that COX-2 mRNA levels are higher in cumulus cells than in mural granulosa cells (3, 12). Therefore, a more detailed analysis of COX-2 mRNA expression before and just after ovulation was undertaken using both RNase protection assays and in situ hybridization.

The results, presented in Figs. 1Go and 2Go, reveal a multiphasic pattern of COX-2 mRNA expression in ovulatory follicles. In cumulus cells a distinctly multiphasic expression pattern, with peaks at 4 and 12 h after hCG treatment, is evident. Mural granulosa cells also show high expression levels at 4 h post-hCG, with expression declining by 8 h. At the 12 h point, mural granulosa cells could not be reliably isolated; therefore, it was not possible to measure COX-2 mRNA levels by RNase protection assay. However, using in situ hybridization, COX-2 mRNA expression appeared to increase in mural granulosa cells between 8 and 12 h after hCG treatment, albeit expression levels remained lower than at 4 h. There was a high degree of consistency between animals in the pattern and level of COX-2 mRNA expression at each time point, indicating that the multiphasic expression patterns identified were not a function of variability in the response to PMSG/hCG treatment. This observation is also supported by the consistent finding that high levels of progesterone receptor mRNA expression were present in the mural granulosa cells of PMSG-treated mice 4 h after hCG treatment, but low levels were present at other times (unpublished results). This timing of PR expression is in line with previous studies (27). Another feature of the COX-2 mRNA expression in ovulatory follicles was the punctate expression pattern. This is most apparent at the 2 and 12 h points in the mural granulosa cell layer, but can also be observed at the 4 and 8 h points (Fig. 2Go). Punctate COX-2 mRNA expression has been observed in mouse ovulatory follicles in other studies (3). The reason for these apparent hot spots of COX-2 mRNA expression is unclear, but may reflect a degree of between-cell heterogeneity in the timing of initiation of COX-2 mRNA expression.



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  		Figure 1. Steady state levels of COX-2 mRNA expression in granulosa cells of preovulatory and ovulatory follicles in mice. Cumulus cells were isolated from PMSG-primed, immature mice at the time of hCG injection (0 h post-hCG) and 2, 4, 8, 12, and 16 h after hCG injection. Mural granulosa cells were isolated from PMSG-primed immature mice 0, 2, 4, and 8 h after hCG injection. Top, Representative phosphorimage (Fuji Photo Film Co., Ltd.) of COX-2 and RPL-19 RNA-RNA-protected hybrids following RNase protection assay and electrophoretic separation of mRNA isolated from cumulus and mural granulosa cell samples. Bottom, Quantified COX-2 mRNA steady state levels in cumulus cells ({square}) and mural granulosa cells ({blacksquare}) after adjusting for between-sample differences in total mRNA concentrations. The values shown are the mean (±SEM) values from four experimental replicates.

 


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  		Figure 2. Localization of COX-2 mRNA in the immature mouse ovary during exogenous gonadotropin-induced preovulatory and ovulatory follicle development. Ovaries from immature, PMSG-primed mice were removed at various times after hCG treatment and fixed in 4% paraformaldehyde. Sections were hybridized overnight with 33P-labeled COX-2 antisense RNA probe. After RNase treatment and washing, slides were exposed to emulsion, developed, and stained with hematoxylin and eosin. The representative sections shown in A–F are brightfield digital images. A, Section of ovary from an immature mouse treated with PMSG 48 h earlier. No hybridization of the COX-2 RNA probe is evident. B–E, Sections of ovary from immature, PMSG-primed mice treated with hCG 2, 4, 8, and 12 h earlier, respectively. Insets in B–D are higher magnification images of individual follicle sections; note the changes in relative COX-2 mRNA expression levels in cumulus and mural granulosa cells. F, Section of ovary and oviduct from immature, PMSG-primed mice treated with hCG 16 h earlier. Inset, COX-2 mRNA expression is evident in cumulus cells in the lumen of the oviduct. Scale bars, approximately 200 µm.

 
As noted above, comparison of COX-2 mRNA expression in mural and cumulus cells 4 h after hCG treatment using the RNase protection assay suggested that the level of COX-2 mRNA expression in these cell types is similar. Despite this, it is apparent from the in situ hybridization results that those mural granulosa cells immediately adjacent to the basement membrane show lower expression levels than the middle and more antral layers of mural granulosa cells. By 8 h after hCG treatment, higher COX-2 mRNA expression levels could be detected in cumulus compared with mural granulosa cells using both the RNase protection assay and in situ hybridization. This difference was also evident at 12 h after hCG treatment. A further noteworthy aspect of the COX-2 mRNA expression pattern found in ovulatory follicles was that 2 h after hCG treatment expression levels were lower in cumulus cells than in mural granulosa cells. Taken as a whole, these data reveal COX-2 mRNA expression to be multiphasic in ovulatory follicles of mice, with substantial changes in the relative expression levels in the different granulosa cell subtypes.

Experiments to investigate the regulation of COX-2 mRNA expression in ovulatory follicles
The expression pattern observed for COX-2 mRNA in ovulatory follicles suggests that the factors regulating expression of this gene may change as the ovulatory process progresses. For this reason, investigation of the factors regulating the expression of this gene was undertaken at three distinct time points. These were the time of the first peak in COX-2 mRNA expression (4 h after hCG treatment), the time of the first nadir in COX-2 mRNA expression (8 h after hCG treatment), and the time of the second peak in COX-2 mRNA expression (12 h after hCG treatment). Depending on the experiment, cells were either isolated from PMSG-primed animals and cultured for 4, 8, or 12 h or were isolated from PMSG-primed, hCG-treated animals and cultured for an additional period, such that the culture period terminated 8 or 12 h after hCG injection.

Regulation of steady-state COX-2 mRNA expression levels in ovulatory follicles 4 h after hCG treatment
In the first series of experiments, regulation of COX-2 mRNA expression at the time of the first peak of expression was examined. Treatment of PMSG-primed mice with 5 IU hCG stimulate COX-2 mRNA expression in both the cumulus and mural granulosa cell compartments of ovulatory follicles in vivo. Therefore, high levels of gonadotropin stimulation appear to be sufficient to initiate COX-2 mRNA expression in these cells in mice. To confirm this, initial trials were undertaken to assess the effects of LH and FSH on COX-2 mRNA expression in mural granulosa cells harvested from PMSG-primed mice and cultured for 4 h. However, expression of COX-2 mRNA was found in control mural granulosa cells cultured for 4 h without either LH or FSH (see Fig. 3Go). Spontaneous changes in highly differentiated granulosa cells have previously been observed in culture (28, 29), with the changes reflecting the in vivo luteinization process found in ovulatory follicles. However, the underlying stimulus for spontaneous luteinization of granulosa cells in vitro is not known. Although there is no evidence to suggest that COX-2 mRNA expression is indicative of luteinization (for example, luteinized granulosa cells are found in the Ptgs2tm1Jedl mouse), it may well be that the cellular basis of spontaneous luteinization in vitro and that of spontaneous COX-2 mRNA expression in vitro are similar. Regardless of whether this is the case, examination of the effect of gonadotropin treatment on the level of COX-2 mRNA expression in cultured mural granulosa cells 4 h after isolation from PMSG-primed mice revealed that both LH and FSH up-regulated mRNA levels. Maximal stimulation with LH occurred at concentrations of 10–500 ng/ml, whereas maximal stimulation with FSH was found at concentrations of 0.1–1.0 IU/ml (full data not shown; see Fig. 3Go).



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  		Figure 3. Gonadotropin regulation of steady state COX-2 mRNA in mural granulosa cells collected from preovulatory follicles and cultured for 4 h. This culture period was timed to coincide with the first COX-2 mRNA expression peak in ovulatory follicles after hCG treatment. Mural granulosa cells isolated from PMSG-primed mice were immediately snap-frozen (first lane) or cultured alone (second lane), with LH (100 ng/ml; third lane), or with FSH (0.5 IU/ml; fourth lane) before being snap-frozen. Note that LH and FSH up-regulated COX-2 mRNA expression; however, untreated cultured mural granulosa cells also (spontaneously) expressed COX-2 mRNA after the period in culture. Data for individual replicates were normalized so that the mean COX-2 mRNA levels for each replicate were equal to 1. Values are expressed as the mean ± SEM. Bars without common letters differ significantly (P < 0.05, at least).

 
To compare the effect of maximally stimulating levels of LH and FSH on COX-2 mRNA levels, mural granulosa cells isolated from PMSG-primed mice were cultured for 4 h alone or with LH (100 ng/ml) or FSH (0.5 IU/ml). As an additional control COX-2 mRNA levels in mural granulosa cells isolated from PMSG-primed mice and immediately snap-frozen were also examined. The results demonstrate the spontaneous expression of COX-2 mRNA in untreated mural granulosa cells cultured for 4 h. Furthermore, the results indicate that maximally stimulating concentrations of LH and FSH promote COX-2 mRNA expression to a similar degree (Fig. 3Go). In the next experiment COX-2 mRNA levels were compared in mural granulosa cells cultured for 4 h in the presence of LH with mural granulosa cells isolated from PMSG-primed mice treated with hCG for 4 h. The results indicate that the level of expression achieved in the cultured cells was lower than that observed in mural granulosa cells stimulated in vivo following hCG treatment (mean ± SEM, 0.46 ± 0.13 vs. 1.53 ± 0.13, respectively; P < 0.01). This difference may reflect subtle differences in the timing of the initiation of COX-2 mRNA expression in the in vivo and in vitro situations. Alternatively, it may be that a factor(s) in addition to high level gonadotropin stimulation is necessary to promote the level of COX-2 mRNA expression observed in vivo in mural granulosa cells 4 h after hCG treatment.

The effects of LH (100 ng/ml) and FSH (0.5 IU/ml) on COX-2 mRNA levels in cumulus cells after 4 h in culture were then examined. COC were isolated from PMSG-primed mice and cultured intact (i.e. with the oocyte still present). Levels of COX-2 mRNA in both the LH-treated and untreated control groups after 4 h of culture were very low (Fig. 4Go). However, FSH initiated high levels of COX-2 mRNA expression in cumulus cells cultured as part of COC. Indeed, expression levels were similar to those found in cumulus cells isolated from mice 4 h after hCG treatment.



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  		Figure 4. Gonadotropin regulation of steady state COX-2 mRNA in cumulus cells after isolation from preovulatory follicles and culture as part of cumulus-oocyte complexes for 4 h. This culture period was timed to coincide with the first COX-2 mRNA expression peak in ovulatory follicles afterhCG treatment. Cumulus-oocyte complexes were isolated from PMSG-primed mice. Oocyte meiotic maturation was inhibited until the start of culture by isolating cumulus-oocyte complexes in medium containing 10 µM milrinone and transfer to medium without milrinone at the start of the culture period. Complexes were cultured alone, with 100 ng/ml LH, or with 0.5 IU/ml FSH before being snap-frozen. In addition, cumulus cells were isolated 4 h after hCG treatment of PMSG-primed mice and immediately snap-frozen. Data for individual replicates were normalized so that the mean COX-2 mRNA levels for each replicate were equal to 1. Values are expressed as the mean ± SEM. Bars without common letters differ significantly (P < 0.05, at least).

 
In the next experiment the role of the oocyte in regulating COX-2 mRNA expression in cumulus cells at the 4 h point was examined. To be able to culture cumulus cells with and without oocytes, COC isolated from PMSG-primed mice were oocytectomized. Cumulus cells (i.e. oocytectomized COC) were then cultured with 0.5 IU/ml FSH, with maturing oocytes, with 0.5 IU/ml FSH plus maturing oocytes, or alone. FSH, but not coculture with oocytes, stimulated COX-2 mRNA expression (Fig. 5AGo). However, high levels of COX-2 mRNA were only found in the presence of both FSH and oocytes.



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  		Figure 5. Oocyte regulation of steady state COX-2 mRNA levels in cumulus and mural granulosa cells after isolation from preovulatory follicles and culture for 4 h. This culture period was timed to coincide with the first COX-2 mRNA expression peak in ovulatory follicles after hCG treatment. The fully grown oocytes used in these experiments were isolated from preovulatory follicles of PMSG-primed mice in medium containing milrinone and then released from inhibition of meiotic maturation at the start of culture by transfer into medium containing no milrinone. A, {square}, Expression levels in cumulus cells isolated from PMSG-primed mice and cultured for 4 h with or without 0.5 IU/ml FSH treatment and with or without oocytes. B, {blacksquare}, Expression levels in mural granulosa cells isolated from PMSG-primed mice and cultured for 4 h with 100 ng/ml LH, with or without oocytes. Cumulus cells were cultured at a concentration of 1.5 oocytectomized cumulus-oocyte complexes/µl; oocytes were cultured at a concentration of 1.5/µl. Data for individual replicates were normalized so that the mean COX-2 mRNA levels for each replicate were equal to 1. Values are expressed as the mean ± SEM. Within a series, bars without common letters differ significantly (P < 0.05, at least).

 
As the levels of COX-2 mRNA found in LH-stimulated cultured mural granulosa cells were lower than those in mural granulosa cells from hCG-treated mice, it may be that a factor(s) in addition to gonadotropin stimulation is necessary to promote appropriate levels of COX-2 mRNA expression in this cell type. Therefore, the possibility that oocytes can promote COX-2 mRNA expression in mural granulosa cells was tested. Mural granulosa cells isolated from PMSG-primed mice were cultured for 4 h with and without maturing oocytes in the presence of 100 ng/ml LH. Coculture with oocytes significantly up-regulated COX-2 mRNA expression in this system (Fig. 5BGo).

Oocytes have previously been shown to have different effects on granulosa cell gene expression depending on the stage of oocyte development. Therefore, in the next experiment the effects of growing, GV stage, and maturing oocytes on COX-2 mRNA expression in FSH-stimulated cumulus cells were compared. All cultures contained 10 µM milrinone, a specific phosphodiesterase-3 inhibitor, to prevent the spontaneous reinitiation of meiosis in GV stage oocytes and to provide experimental balance in the other groups. Maturing oocytes were cultured without milrinone for 1.5 h before the experiment to allow these oocytes to undergo germinal vesicle breakdown. The results show that growing oocytes had no effect on COX-2 mRNA expression, whereas both GV stage and maturing oocytes promoted COX-2 mRNA levels to a similar degree (Fig. 6Go).



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  		Figure 6. Effect of the developmental stage of the oocyte on cumulus cell COX-2 mRNA expression. Cumulus-oocyte complexes were isolated from PMSG-primed mice, oocytectomized, and cultured for 4 h in the presence of 0.5 IU/ml FSH alone, with growing oocytes, with fully grown GV stage oocytes, or with fully grown meiotically maturing oocytes. Cumulus cells were cultured at a concentration of 1.5 oocytectomized cumulus-oocyte complexes/µl; growing oocytes were isolated from preantral follicles and were cultured at a concentration of 3/µl; fully grown GV stage and meiotically maturing oocytes were isolated from preovulatory follicles and were cultured at a concentration of 1.5/µl. All groups contained 10 µM milrinone to inhibit meiotic maturation of fully grown GV stage oocytes and to provide experimental balance in the other groups. Maturing oocytes were precultured for 1.5 h in milrinone-free medium to allow the maturation process to initiate. Data for individual replicates were normalized so that the mean COX-2 mRNA levels for each replicate were equal to 1. Values are expressed as the mean ± SEM. Bars without common letters differ significantly (P < 0.05, at least).

 
Regulation of steady-state COX-2 mRNA expression levels in ovulatory follicles 8 and 12 h after hCG treatment
The initial in vivo survey of the kinetics of COX-2 mRNA expression after hCG treatment showed a biphasic pattern, with peaks 4 and 12 h after hCG treatment in cumulus cells. However, this pattern of cumulus cell expression of COX-2 mRNA was not observed when COC were cultured in the presence of 0.5 IU/ml FSH for up to 14 h (Fig. 7Go). In this situation, whereas COX-2 mRNA expression declined by 8 h, no second peak of expression could be identified. Similar results were observed when COC were isolated from mice 4 h after hCG treatment and cultured for 4–10 h in the absence of FSH (i.e. 8–14 h after hCG treatment; data not shown). Furthermore, no second peak in expression was observed when COC were cultured in the presence of LH (100 ng/ml) or without gonadotropin stimulation (data not shown). These results suggest that the second peak in COX-2 mRNA expression may not be under (direct) gonadotropic control in cumulus cells.



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  		Figure 7. This experiment examined the pattern of COX-2 mRNA expression in cumulus cells cultured for up to 14 h as part of FSH-stimulated cumulus-oocyte complexes. Cumulus-oocyte complexes were isolated from PMSG-primed mice and cultured in the presence of 0.5 IU/ml FSH for 4–14 h. Note the absence of a second peak in COX-2 mRNA expression using this experimental system. Data for individual replicates were normalized so that the mean COX-2 mRNA levels for each replicate were equal to 1. Values are expressed as the mean ± SEM.

 
One reason for the failure of the second peak of COX-2 mRNA expression in vitro may be a lack of appropriate stimulation from a factor(s) derived from the follicle wall. To test this hypothesis, COC were cocultured with or without follicle shells (principally thecal and mural granulosa cells) between 8 and 12 h after hCG treatment. Both COC and follicle shells were isolated from mice 8 h after hCG treatment. Using this system, cumulus cell expression of COX-2 mRNA was significantly higher than that when COC were cultured alone (Fig. 8Go). Indeed, COX-2 mRNA levels in COC cocultured with follicle shells did not differ from those in cumulus cells isolated from mice 12 h after hCG treatment. These results therefore support the hypothesis that the second COX-2 peak after hCG treatment is dependent upon stimulation from a follicle wall-derived factor.



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  		Figure 8. This experiment examined the role of follicular-derived factors in the regulation of cumulus cell COX-2 mRNA expression in ovulatory follicles 12 h after hCG treatment. This culture period was timed to coincide with the second peak in COX-2 mRNA expression in ovulatory follicles after hCG treatment. To test the role of follicular-derived factors in regulating this expression, cumulus-oocyte complexes were cocultured with or without follicle shells between 8 and 12 h after hCG treatment. Cumulus cells were then isolated and snap-frozen before assay for COX-2 mRNA content. In both groups, follicle shells and cumulus-oocyte complexes were isolated from PMSG-primed mice treated with hCG for 8 h. Cumulus-oocyte complexes were cultured at 1.5 complexes/µl and follicle shells at 0.2 shells/µl. As a further control, cumulus cells were isolated from PMSG-primed mice 12 h after hCG treatment and immediately snap-frozen. Data for individual replicates were normalized so that the mean COX-2 mRNA levels for each replicate were equal to 1. Values are expressed as the mean ± SEM. Bars without common letters differ significantly (P < 0.05, at least).

 
To try to identify this factor, COC from PMSG-treated mice were cultured for 12 h in the presence of FSH (0.5 IU/ml). Groups were treated with tumor necrosis factor-{alpha}, IL-1ß, IL-6, insulin-like growth factor I, EGF, or PGE-2. Each of these agonists has been implicated in regulating the ovulatory process or in cumulus cell function [see, for example, tumor necrosis factor-{alpha} (30), IL-1ß (31), IL-6 (32), insulin-like growth factor I (33), EGF (34), and PGE2 (35)]. Treatments were applied after 8 h. Using this experimental system, only IL-1ß stimulated COX-2 mRNA expression (Fig. 9Go). Similarly, only IL-1ß stimulated COX-2 mRNA expression levels when COC were isolated from ovulatory follicles 8 h after hCG treatment and then cultured for 4 h in the absence of FSH. As IL-1ß is thought to be expressed in thecal and mural granulosa cells at late stages of ovulation (31) it was hypothesized that IL-1ß is the follicular-derived factor that stimulates the second peak of COX-2 mRNA expression in cumulus cells. To test this, COC and follicle shells isolated from mice 8 h after hCG treatment were cocultured for 4 h in the presence or absence of 200 ng/ml IL-1Ra. This antagonist blocks the action of IL-1ß by binding to the IL-1 type 1 receptor (36). In preliminary trials 200 ng/ml IL-1Ra fully blocked the action of 0.1 ng/ml IL-1ß on COX-2 mRNA expression in 12-h cultures of FSH-stimulated COC isolated from PMSG-treated mice (data not shown). However, in the COC/follicle coculture system there was no effect of IL-1Ra on COX-2 mRNA expression in cumulus cells (mean ± SEM relative levels of COX-2 mRNA in control and IL-1Ra-treated cumulus cells, 0.97 ± 0.02 and 1.03 ± 0.02, respectively; P > 0.1). This suggests that IL-1ß is not a physiological stimulator of the second COX-2 mRNA expression peak in cumulus cells. Alternatively, IL-1ß may not act through the IL-1 type 1 receptor to stimulate COX-2 mRNA expression in cumulus cells.



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  		Figure 9. Effect of IL-1ß on steady state expression of COX-2 mRNA in cumulus cells isolated from PMSG-primed mice and cultured as part of cumulus-oocyte complexes for 12 h. This culture period was timed to coincide with the second peak in COX-2 mRNA expression in ovulatory follicles after hCG treatment. Cumulus-oocyte complexes were cultured at a concentration of 1.5/µl for 12 h in the presence of 0.5 IU/ml FSH. Groups were treated with 0–1.0 ng/ml IL-1ß, with this treatment applied after 8 h of culture. Data for individual replicates were normalized so that the mean COX-2 mRNA levels for each replicate were equal to 1. Values are expressed as the mean ± SEM. Bars without common letters differ significantly (P < 0.05, at least).

 
In the final series of experiments, the likely role of the oocyte in promoting changes in COX-2 mRNA expression at 8 and 12 h post-hCG was assessed. To examine the role of the oocyte at 8 h post-hCG, COC were isolated from mice 4 h after hCG treatment and oocytectomized. Cumulus cells were then cultured with or without maturing oocytes between 4 and 8 h after hCG treatment. In this system the presence of oocytes significantly up-regulated COX-2 mRNA expression (Fig. 10AGo). Similar results were observed when COC were isolated from primed mice and treated with 0.5 IU/ml FSH for 4 h before oocytectomy and coculture with and without oocytes. To examine the role of the oocyte in regulating COX-2 mRNA expression 12 h after hCG, COC, maturing oocytes, and follicle shells were isolated from hCG-treated mice 8 h after injection, and the COC were oocytectomized. For a 4-h period, cumulus cells were cultured with follicle shells to promote COX-2 mRNA expression levels and cocultured with or without maturing oocytes. The results show that oocytes promote COX-2 mRNA expression at 12 h (Fig. 10BGo).



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  		Figure 10. Role of the oocyte in regulating cumulus cell COX-2 mRNA expression in ovulatory follicles 8 and 12 h after hCG treatment. These culture periods were timed to coincide with the nadir in COX-2 expression in ovulatory follicles (8 h) and the second peak in COX-2 mRNA expression in ovulatory follicles. A, Examination of the role of the oocyte in regulating cumulus cell COX-2 mRNA expression 8 h after hCG treatment. Cumulus cells were isolated from PMSG-primed mice 4 h after hCG treatment and cultured for an additonal 4 h in the presence or absence of fully grown, meiotically maturing oocytes also isolated from PMSG-primed mice 4 h after hCG treatment. B, Examination of the role of the oocyte in regulating cumulus cell COX-2 mRNA expression 12 h after hCG treatment. Cumulus cells, follicle shells, and fully grown, meiotically maturing oocytes were isolated from PMSG-primed mice 8 h after hCG treatment. Cumulus cells were then cultured for an additional 4 h in the presence of 0.2 follicle shells/µl with or without oocyte coculture. Follicle shells were included in the culture because previous results indicated that coculturing cumulus-oocyte complexes with these shells between 8 and 12 h after hCG treatment promoted cumulus cell COX-2 mRNA expression to a level found in ovulatory follicles 12 h after hCG treatment. However, follicle shells were not included in A because at this time point cumulus cells cultured in the absence of follicle shells display a similar level of COX-2 mRNA expression as cumulus cells isolated from ovulatory follicles 8 h after hCG treatment (data not shown). Data for individual replicates were normalized so that the mean COX-2 mRNA levels for each replicate were equal to 1. Values are expressed as the mean ± SEM. Within a series, bars without common letters differ significantly (P < 0.05, at least).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report documents the pattern of COX-2 mRNA expression in granulosa cells of ovulatory follicles and examines the regulation of the multiphasic expression of the Ptgs2 gene in mice. The principal focus of the study was to determine the importance of the oocyte in regulating COX-2 mRNA expression and to use this information to evaluate the role of the oocyte in the ovulatory process. It has previously been suggested that oocytes regulate the expression of COX-2 because GDF-9, an oocyte-specific secreted factor in the ovary, promotes COX-2 mRNA expression in mural granulosa cells (18, 21). Furthermore, mural granulosa and cumulus cells exhibit divergent levels of COX-2 mRNA expression, supporting this concept. The results suggest that the oocyte resident in a mouse ovulatory follicle promotes expression of COX-2 mRNA, but does not orchestrate the multiphasic changes in COX-2 mRNA levels observed in these follicles.

The central finding of this study is that coculture of fully grown oocytes with either cumulus or mural granulosa cells promotes the expression of COX-2 mRNA. This action of fully grown oocytes was evident at all time intervals examined, supporting the hypothesis that the oocyte resident in an ovulatory follicle promotes the expression of the Ptgs2 gene. Expression of Ptgs2 (COX-2) mRNA may be higher in cumulus cells than in mural granulosa cells 8 and 12 h after hCG treatment because of the proximity of cumulus cells to the oocyte. In this scenario, a concentration gradient of oocyte-derived stimulatory factor(s) is established within the follicle, with the cumulus cells receiving higher levels of stimulation and therefore expressing more COX-2 mRNA than the mural granulosa cells. However, as mural granulosa cells and cumulus cells express similar levels of COX-2 mRNA 4 h after treatment with hCG, it is likely that factors other than the oocyte promote the expression of COX-2 mRNA in mural granulosa cells during the early stages of the ovulatory process. As mural granulosa cells from preovulatory follicles express receptors for LH and express COX-2 after treatment with hCG in vivo and with LH in vitro, it is probable that LH acts directly on mural granulosa cells. Studies in rats support this idea, showing that both forskolin, an activator of adenylyl cyclase, and LH, which also acts via the cAMP pathway, up-regulate COX-2 expression in cultured granulosa cells (37). It therefore seems likely that LH is the principal determinant of levels of COX-2 mRNA in mural granulosa cells during the first peak of expression in ovulatory follicles.

This is not to say that a role for the oocyte in promoting the expression of COX-2 mRNA in mural granulosa cells can be ruled out. Indeed, expression is higher in mural granulosa cells closest to the antrum, suggesting that the oocyte may play an important role in promoting COX-2 mRNA expression in these cells. This conclusion is supported by the observations that expression of COX-2 mRNA increases in mural granulosa cells in response to oocyte-derived factors during coculture with oocytes, and that expression of COX-2 mRNA is higher in mural granulosa cells in vivo than after stimulation with LH in vitro. Therefore, the effects of LH and oocyte-secreted factors probably account for the heterogeneous pattern of expression of COX-2 in mural granulosa cells.

Just as mural granulosa COX-2 mRNA expression levels may be the result of stimulation from both oocyte-derived and non-oocyte-derived factors, factors other than those supplied by the oocyte are essential determinants of COX-2 mRNA expression levels in cumulus cells. This observation is supported by the finding that oocyte-derived factors are necessary, but not sufficient, to initiate high levels of COX-2 mRNA expression in cumulus cells in vitro. The identity of the non-oocyte-derived factor(s) necessary for the initiation of COX-2 mRNA expression in cumulus cells is unclear. In vivo, cumulus cell expression of COX-2 mRNA is initiated after hCG treatment. However, mouse cumulus cells do not express detectable levels of LH receptor mRNA (13) and do not initiate COX-2 mRNA expression in response to LH treatment in vitro. This indicates that the action of hCG treatment on cumulus cells in vivo is probably mediated by an intermediary signal. Presumably, this signal is generated or made active as a consequence of hCG stimulation of mural granulosa, or possibly thecal, cells. It is interesting to speculate that the lower level of COX-2 mRNA expression in cumulus compared with mural granulosa cells 2 h after hCG treatment is a reflection of the time required to generate the signal that initiates COX-2 mRNA expression in cumulus cells.

Identification of the factor(s) responsible for initiating COX-2 mRNA expression in cumulus cells is an important objective for future studies. In the current study, experiments examining the role of the oocyte in cumulus cell COX-2 mRNA expression at the 4 h point took advantage of the fact that 0.5 IU/ml FSH promoted COX-2 mRNA expression in cultured cumulus-oocyte complexes. As hCG treatment alone is sufficient to stimulate COX-2 mRNA expression in cumulus cells in vivo, such high levels of FSH stimulation are not necessary for COX-2 mRNA expression to occur. However, in the absence of information about the physiological signal(s) necessary to initiate COX-2 mRNA expression in cumulus cells, the use of FSH treatment to stimulate COX-2 mRNA expression in vitro is supported by a number of observations. Firstly, the concentration of FSH used stimulates cumulus expansion in vitro (our unpublished observations), indicating that the treatment promotes not just COX-2 mRNA expression but a range of functional changes typical of cumulus cells in ovulatory follicles. Secondly, as a consequence of FSH treatment, cumulus cells cultured for 4 h as intact cumulus-oocyte complexes exhibited COX-2 mRNA expression levels similar to those of cumulus cells at the first expression peak in vivo. This indicates that cumulus cells express physiologically appropriate levels of COX-2 mRNA after FSH treatment. Thirdly, the response of cumulus cells to oocyte coculture at the 8 and 12 h points was similar whether COX-2 mRNA expression had been initiated in vitro using FSH or in vivo using hCG treatment. Therefore, at these time intervals the mode of initiation of COX-2 mRNA expression in cumulus cells had no effect on the subsequent response of these cells to oocyte coculture. These findings therefore support the use of FSH treatment as a model to examine the role of the oocyte in regulating the first peak in COX-2 mRNA expression in cumulus cells.

Evidence was also found of a role for non-oocyte-derived factors in promoting the second COX-2 mRNA expression peak. Importantly, cultured cumulus-oocyte complexes failed to exhibit a second peak of expression in the absence of additional stimulation. Furthermore, coculture of follicular shells with cumulus-oocyte complexes between 8 and 12 h after hCG treatment stimulated cumulus cell COX-2 mRNA expression to in vivo levels. There is, therefore, likely to be a follicle wall-derived factor(s) that is necessary for stimulating the second peak in COX-2 mRNA expression in cumulus cells. Limited attempts to identify this factor indicated that IL-1ß may be a candidate because cumulus cells showed higher steady state levels of COX-2 mRNA when this factor was included in the culture system between 8 and 12 h after hCG treatment. This finding complements previous evidence from the rat that IL-1ß can induce COX-2 expression in ovarian cells within 1 h of treatment (38). Furthermore, localization studies indicate that IL-1 receptor expression is initiated in cumulus cells during the periovulatory period, and thecal cells express IL-1ß at this time in the mouse (31). However, the possibility that IL-1ß may be the follicle wall-derived factor that up-regulates COX-2 mRNA expression in cumulus cells at the 12 h point was not supported by experiments in this report using IL-1Ra. These experiments showed that IL-1Ra effectively blocked IL-1ß action in vitro, but had no effect on COX-2 mRNA expression when cumulus-oocyte complexes were cocultured with follicle shells. Furthermore, IL-1ß-deficient female mice have normal fertility and therefore do not phenocopy Ptgs2tm1Jedl mice (39). Whatever the identity of the follicle wall-derived factor that up-regulates cumulus cell COX-2 mRNA levels, it is clear that the oocyte augments the stimulatory action of this factor in vitro. COX-2 mRNA levels in cumulus cells 12 h after hCG treatment are therefore likely to be a function of both an oocyte-derived factor and a factor derived from the follicle wall.

Eight hours after hCG treatment there is little evidence of a role for non-oocyte-derived factors in regulating COX-2 mRNA levels in cumulus cells, as the decline in COX-2 mRNA expression evident in cultured cumulus-oocyte complexes at this time is similar to that found in cumulus cells in vivo. Oocyte coculture promoted COX-2 mRNA expression in cumulus cells at 8 h after hCG treatment, suggesting that the decline in COX-2 mRNA levels occurs despite the activity of the oocyte and is not caused by a factor(s) suppressing COX-2 mRNA expression, unless this factor is cumulus derived.

Overall, the evidence from this study strongly supports the concept that the oocyte resident within mouse ovulatory follicles produces a factor(s) that promotes the expression of COX-2 mRNA. It is notable that the production of these factor(s) by fully grown oocytes occurs throughout the ovulatory process and is probably not dependent on meiotic status. This indicates that oocytes have a role in promoting COX-2 mRNA expression throughout the ovulatory process in mice, but are not important in orchestrating the multiphasic changes that occur in COX-2 mRNA expression in ovulatory follicles. Instead, the timing of these changes probably results in the first instance from the LH surge. Constitutive, rather than regulated, production of factors that promote COX-2 mRNA expression by the oocyte may well function to support the ovulatory process while allowing ovulationassociated events both near and outside the vicinity of the oocyte to occur in a coordinated fashion. This conclusion is also of interest because of species differences in the timing of initiation of COX-2 expression during the ovulatory process (1, 2, 40, 41). The current results do not suggest that the oocyte is likely to play a role in determining these differences.

As growing oocytes do not promote COX-2 mRNA expression, the ability to produce this factor(s) appears to be developmentally regulated. Acquisition of this ability by oocytes is therefore likely to be a prerequisite for the appropriate differentiation of cumulus cells, and possibly mural granulosa cells, in ovulatory follicles after the LH surge. It is not known whether the ability of the oocyte to promote COX-2 mRNA expression is essential to ovulation. The anovulatory phenotype of the Ptgs2tm1Jed mouse (COX-2 knockout) is good evidence that the expression of this gene per se is essential for ovulation. However, it may be that deficiencies in aspects of the ovulatory process independent of oocyte regulation disrupt ovulation in these mice. On the other hand, it has been shown that Ptgs2tm1Jed mice exhibit defective cumulus expansion (42), suggesting that the expression of COX-2 in cumulus cells may be at least a component of the anovulatory phenotype of the Ptgs2tm1Jed mouse. This idea is supported by experiments showing that appropriate cumulus expansion is important for ovulation (43). In this situation, it is possible that the developmentally regulated acquisition of COX-2 mRNA-promoting activity by oocytes may represent a mechanism to reduce the chances of immature or developmentally incompetent oocytes from being ovulated.

The identity of the oocyte-derived factor(s) that promotes COX-2 mRNA expression is not known with certainty, although it has many of the features of the as yet uncharacterized cumulus expansion-enabling factor. For example, both cumulus expansion-enabling factor and the COX-2 mRNA expression-promoting factor are produced by fully grown, but not growing, oocytes (44). Furthermore, both factors appear to be produced by fully grown oocytes regardless of meiotic status (44). As recombinant GDF-9 promotes COX-2 mRNA expression in both mural granulosa and cumulus cells (19, 22) (our unpublished results), this oocyte-specific secreted protein is a candidate for the COX-2 mRNA expression-promoting factor. On the other hand, production of GDF-9 is essential to preantral follicle development (17), but growing oocytes do not promote cumulus cell COX-2 mRNA expression in vitro. These apparently contradictory results are the subject of ongoing investigations in our laboratory.

In conclusion, the results of this study indicate that the oocyte resident within ovulatory follicles produces a factor(s) that promotes the expression of COX-2 mRNA by cumulus cells and possibly by mural granulosa cells in mice. However, the dynamic multiphasic changes in the expression of COX-2 mRNA appear to be orchestrated by non-oocyte-derived factors. As the expression of COX-2 is important for ovulation, the results suggest that the oocyte may play a crucial role in supporting the ovulatory process.


   Acknowledgments
 
We are grateful to Dr. S. K. Dey for the COX-2 complementary DNA, to R & D Systems, Inc., for the gift of the interleukin-1 receptor antagonist, to Organon for the gift of the FSH, and to Dr. A. Parlow, NIDDK, for the gifts of LH and PMSG. We thank Drs. Wes Beamer and Robert Taft for their helpful comments during manuscript preparation. The authors are very grateful to Priscilla Jewett and Gregory Martin of Biological Imaging Service and Jennifer Smith, of Multi Media Services for their assistance in preparing the histology samples and photomicrographs.


   Footnotes
 
1 This work was supported by the NICHHD Grant HD-23839. The scientific services of The Jackson Laboratory receive support from a Cancer Center Core Grant (CA34196) the NCI. Back

2 Present address: School of Biology, University of Leeds, Leeds, United Kingdom LS2 9JT. Back

Received January 9, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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J. Kim, M. Sato, Q. Li, J. P. Lydon, F. J. DeMayo, I. C. Bagchi, and M. K. Bagchi
Peroxisome Proliferator-Activated Receptor {gamma} Is a Target of Progesterone Regulation in the Preovulatory Follicles and Controls Ovulation in Mice
Mol. Cell. Biol., March 1, 2008; 28(5): 1770 - 1782.
[Abstract] [Full Text] [PDF]

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 Hum Reprod UpdateHome page
R. B. Gilchrist, M. Lane, and J. G. Thompson
Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality
Hum. Reprod. Update, March 1, 2008; 14(2): 159 - 177.
[Abstract] [Full Text] [PDF]

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