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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
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-
(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 [-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
[-32P]CTP (NEN Life Science Products). [-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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results, presented in Figs. 1
and 2, 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. 2). 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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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. 3). 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. 3).
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). 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. 4). 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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). However, high
levels of COX-2 mRNA were only found in the presence of both FSH and
oocytes.
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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. 6).
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). 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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). 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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, 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-
(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. 9). 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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). 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. 10B).
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Discussion |
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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.
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2 Present address: School of Biology, University of Leeds, Leeds,
United Kingdom LS2 9JT.
Received January 9, 2001.
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(TNF) on ovulation in the rat
ovary. Reprod Fertil Dev 7:67–73[CrossRef][Medline]
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) and mural granulosa cells (
) after adjusting for
between-sample differences in total mRNA concentrations. The values
shown are the mean (±SEM) values from four experimental
replicates.
