Timely interaction between prostaglandin andchemokine signaling is a prerequisite forsuccessful fertilization
Shigero Tamba*†, Rieko Yodoi*†, Eri Segi-Nishida*‡, Atsushi Ichikawa*§, Shuh Narumiya‡, and Yukihiko Sugimoto*¶*Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, and ‡Department of Pharmacology, Faculty of Medicine,
Kyoto University, Kyoto, Japan
Edited by Salvador Moncada, University of London, London, United Kingdom, and approved July 17, 2008 (received for review June 12, 2008)

cumulus cells 兩 extracellular matrix 兩 fibronectin 兩 integrin 兩
prostanoid receptor EP2

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O

vulation and fertilization are key processes in mammalian
female reproduction, which are strictly regulated by pituitary
gonadotropins, follicle-stimulating hormone, and luteinizing hormone. These hormones induce various preovulatory processes,
including follicular development, oocyte maturation, and cumulus
expansion, and trigger ovulation (1). Ovulated eggs move to theoviduct; subsequently the timely interaction between an egg andsperm leads to successful fertilization (2). Whereas various molecules are known to regulate preovulatory processes locally in thefollicle under the control of gonadotropin (3), little is known about
the regulation of postovulatory processes, including fertilization in
the oviduct, which gonadotropin does not control.
One potential candidate for the local mediators regulating fertilization within the oviduct is chemokines. Chemokines constitutea family of structurally related, inducible peptides involved invarious immune responses and inflammation, acting as chemoattractants and activators of specific types of leukocytes (4). Approximately 40 chemokines, grouped into distinct families and secreted
by various cells, are currently known (5). It was recently reported
that in humans, CCL5 (also called RANTES) is present in thefollicular fluid and stimulates the in vitro migration of sperm, which
express CCR1, a receptor for CCL5 (6). Based on these findings, it
has been proposed that chemokines may participate in sperm
guidance within the genital tract (7). The physiological significanceof such chemokine signaling within the oviduct remains elusive,
however.
The cumulus oophorus is composed of a group of closely
associated granulosa cells that surround the oocyte in the antral
follicle and are collectively called cumulus cells (8). In response toa surge in luteinizing hormone, the cumulus cells start to produce
extracellular matrix (ECM) components, which are deposited into
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0805699105

the intercellular space and are stabilized by accessory proteins. Thisphenomenon is called cumulus expansion. A major component of
the ECM produced by the cumulus cells is hyaluronan, which
provides the viscoelastic properties of the cumulus oophorus. The
expanded cumulus oophorus (cumulus cells and ECM) makes atight complex with an oocyte that is ovulated as the cumulus-oocyte
complex (COC). During ovulation, the cumulus oophorus protects
the oocyte from mechanical stress and from proteolytic enzymes
present in the follicle and oviduct, and directs the oocyte into theoviduct by facilitating its capture by the ciliated epithelial cells of
the infundibulum and its transport to the fertilization site (9). In theoviduct, the cumulus oophorus facilitates access of the sperm to the
oocyte by trapping and selecting sperm for successful fertilization(10). Thus, the complex organization of the oocyte, cumulus cells,and ECM is essential for successful fertilization in the oviduct.
Indeed, recent studies using mice null of several hyaluronan-binding
proteins have shown that the cumulus ECM is required for successful fertilization in vivo; female mice deficient in these molecules
are sterile, due to loss of the cumulus and ECM (11–13). However,once sperm reach the COC, the cumulus ECM must be disassembled so that the sperm can find a passage through the ECM layer.
Although it has been suggested and is generally believed that the
cumulus ECM is disassembled by sperm motility and sperm hyaluronidase (14), whether the disassembly of the cumulus ECM is
induced by the force of the sperm alone or is regulated in an
autocrine or paracrine manner by the cumulus itself remains
unclear.
Prostaglandin E2 (PGE2), an arachidonate metabolite synthesized most abundantly within the follicle, is a key mediator in theovulation-stimulating action of gonadotropin (15). PGE2 exerts its
actions by acting on four subtypes of the PGE receptor, EP1–EP4(16). On gonadotropin stimulation, cyclooxygenase-2 (COX-2;
Ptgs2), a rate-limiting enzyme of PG biosynthesis, is induced in allcells within the follicle (17), and a large amount of PGE2 is released
into the follicular fluid (15). Indeed, Ptgs2 null mice demonstrate
severely impaired ovulation (18). Similarly, mice lacking the PGE
receptor EP2 (Ptger2), which is expressed in the cumulus cells,
exhibit reduced ovulation (19). These findings clearly indicate that
PGE2-EP2 signaling in the cumulus plays a role in the ovulatory
Author contributions: S.T., E.S.-N., A.I., S.N., and Y.S. designed research; S.T. and R.Y.
performed research; S.T., R.Y., and Y.S. analyzed data; and S.T., R.Y., E.S.-N., S.N., and Y.S.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
†S.T. and R.Y. contributed equally to this work
§Present address: School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s

University, Nishinomiya, Hyogo 663-7189, Japan¶To whom correspondence should be addressed at: Department of Physiological Chemistry,

Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501,
Japan. E-mail: ysugimot@pharm.kyoto-u.ac.jp.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0805699105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA

PNAS 兩 September 23, 2008 兩 vol. 105 兩 no. 38 兩 14539 –14544

MEDICAL SCIENCES

Timely interaction between the egg and sperm is required for successful fertilization; however, little is known about the signaling
therein. Prostaglandin (PG) E receptor EP2-deficient (Ptger2ⴚ/ⴚ) female mice exhibit a severe fertilization defect. We investigated themolecular events leading to this failure. We found increased gene
expression for chemokines, such as Ccl2, Ccl7, and Ccl9, in Ptger2ⴚ/ⴚcumulus cells (the somatic cells surrounding the egg) compared withwild-type cells. Furthermore, under physiological conditions, cumulus-derived chemokine signaling was found to have a dual action;
CCL7 facilitates sperm migration to the cumulus– egg complex andintegrin-mediated cumulus extracellular matrix (ECM) assembly toprotect eggs. However, in the absence of PGE2-EP2 signaling, chronic
CCL7 signaling results in excessive integrin engagement to the ECM,
making the cumulus ECM resistant to sperm hyaluronidase, thereby
preventing sperm penetration. Our findings indicate that PGE2-EP2
signaling negatively regulates the autocrine action of chemokinesand prevents excessive cumulus ECM assembly. This interaction
between PG and chemokine signaling is required for successful
fertilization.

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mice exhibit impaired fertilization and abortive expansion of the
cumulus after ovulation; however, why Ptger2 deficiency leads to asevere reduction in fertilization rate was unclear (19). Because
Ptger2 is induced in cumulus cells on gonadotropin stimulation, we
hypothesized that the PGE2-EP2 signaling in cumulus cells regulates the expression of unidentified molecules with important
regulatory roles in fertilization. To identify such key molecules, weused oligonucleotide arrays as a screening tool. COCs were collected from the oviducts of WT and Ptger2⫺/⫺ mice 14 h after
injection of human CG (hCG) and were subjected to analysis after
oocyte removal. In the initial screen of the ⬇12,000 genes represented on the array, we identified 16 genes showing more than afivefold increase and 7 genes showing more than a fivefold decreasein expression in the Ptger2⫺/⫺ cumulus cells compared with the WT
cumulus cells [supporting information (SI) Table S1]. Among the 7
genes down-regulated in the Ptger2⫺/⫺ cumulus cells, the Ptger2
gene showed the greatest reduction, indicating that the expression
analysis was working accurately. Among the 16 genes up-regulated
in the Ptger2⫺/⫺ cumulus cells, we found 4 genes for chemokine
ligands, Ccl2 (MCP-1), Ccl9 (MIP-1␥), Ccl7 (MCP-3), and Cxcl1(Gro␣), along with 2 genes for chemokine receptors, Ccr5 (CCR5)
and Ccr1 (CCR1). We also detected significantly enhanced expression of Ccr2 (CCR2) (Table S1). Because a role for chemokines in
fertilization has not been well established, we focused on the three
aforementioned C-C chemokines (CCL2, CCL7, and CCL9) and
explored the possibility that these chemokines may regulate fertilization in an autocrine manner.
We first addressed the time course of gene expression of thechemokines and CCRs in both WT and Ptger2⫺/⫺ cumuli. Cumuli
from five animals were pooled for each time point and subjected toreal-time reverse transcription–polymerase chain reaction (RTPCR) (Fig. 1A). Before ovulation (at 3 and 9 h after hCG injection),
the expression levels of Ccl9 mRNA were as low as the backgroundlevels in both genotypes; however, Ccl9 expression in the WT
cumuli was increased by fivefold at 14 h, when the COCs moved tothe oviduct. Moreover, the induction of Ccl9 expression on ovulation was augmented significantly in EP2⫺/⫺ cumuli. Similar results
were obtained for the expression patterns of Ccl2, Ccl7, Ccr1, Ccr2,
and Ccr5 in the cumulus cells. In contrast, no expression of any
chemokine genes was detected in the WT and Ptger2⫺/⫺ oocytes(data not shown). These results indicate that chemokine genes are
induced in the cumuli on ovulation, and that this induction isenhanced in Ptger2⫺/⫺ cumuli. Indeed, a statistically significant
14540 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0805699105

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processes. Notably, however, Ptger2⫺/⫺ mice show a higher rate of
failure in fertilization than in ovulation; the ovulation number ofPtger2⫺/⫺ mice is 80% of that of wild-type (WT) mice, whereas thefertilization rate of Ptger2⫺/⫺ mice is ⬇20% of that of WT mice.
Both the Ptgs2 and Ptger2 genes are still highly expressed in cumulus
cells even after ovulation. Intriguingly, COCs isolated from thePtger2⫺/⫺ oviduct show a significant reduction in in vitro fertilization
with sperm compared with control WT COCs (19), suggesting that
PGE2-EP2 signaling contributes to fertilization-related processes,
such as cumulus ECM disassembly and sperm penetration.
To gain insight into the mechanism causing fertilization failure in
the Ptger2⫺/⫺ cumulus, in this study we compared the gene expression profiles of WT and Ptger2⫺/⫺ cumuli isolated from the oviduct.
We found increased gene expression for chemokines, such as Ccl2,
Ccl7, and Ccl9, in Ptger2⫺/⫺ cumuli compared with WT cumuli. We
further examined the physiological function of cumulus-derived
chemokines in postovulatory processes and found that the chemokines facilitate both sperm attraction and compaction of the
cumulus by integrin-mediated ECM assembly, the latter of which is
down-regulated by PGE2-EP2 signaling to allow sperm penetration
for successful fertilization.

Fig. 1. Expression of chemokine genes in ovulated cumuli and its enhancement by EP2 deficiency. (A) Time course of chemokine gene expression in WT(open circles) and Ptger2⫺/⫺ (KO; gray squares) cumulus cells. Expression levels
are presented as fold change of the values at 3 h. (B) Increased expression of
Ccl9 and Ccl7 in the cumuli isolated from Ptger2⫺/⫺ and indomethacin-treatedmice. The cumuli were collected individually from WT, Ptger2⫺/⫺ (KO), vehicletreated (Cont), and indomethacin-treated mice (Indo) at 14 h, and the RNA
from each mouse was subjected to real-time RT-PCR analysis. Values arepresented as mean ⫾ standard error of the mean (SEM) (n ⫽ 3–5). **, P ⬍ 0.01.
(C) Enhanced expression of CCL2 protein in oviductal cumuli in Ptger2⫺/⫺ mice.
Bright-field and immunofluorescence images showing signals for CCL2 protein in the cumuli from WT and Ptger2⫺/⫺ (KO) mice. Scale bar: 20 ␮m. (D) PGE2
attenuates and indomethacin augments ccl gene expression in WT, but not in
Ptger2⫺/⫺ COCs. COCs collected from WT and Ptger2⫺/⫺ (KO) mice wereincubated with 1 ␮M PGE2, 2 mM dibutyryl cAMP (cAMP), 10 ␮M indomethacin(Indo), or vehicle (Cont) for 3 h. Total RNA was extracted and subjected toreal-time RT-PCR analysis. Expression levels are shown as fold change of thelevel of WT control. Values are presented as mean ⫾ SEM (n ⫽ 3). *, P ⬍ 0.05;**, P ⬍ 0.01. Representative results of two (A and D) or three (B and C)
independent experiments are shown.

difference in Ccl9 and Ccl7 gene expression was detected between
WT and Ptger2⫺/⫺ cumuli at 14 h, when the cumulus RNA isolated
from each animal was individually subjected to real-time RT-PCR(Fig. 1B).
To examine the protein expression of chemokines in the cumuli,
we prepared ovary sections (at 3 and 9 h) and oviductal ampulla
sections (at 14 h) from WT and Ptger2⫺/⫺ animals and subjected
them to immunofluorescence analysis for CCL2, CCL7, and CCL9.
Whereas immunofluorescence signals for these chemokines were atbackground levels in both WT and Ptger2⫺/⫺ ovarian tissues before
ovulation (data not shown), weak but specific signals for CCL2 weredetected in the WT cumuli after ovulation (i.e, at 14 h) (Fig. 1C).
Notably, these CCL2 signals were stronger in the Ptger2⫺/⫺ cumuli
than in the WT cumuli. Similar results were obtained for CCL7 andCCL9 (data not shown).
To verify that a loss of PG signaling around the time of ovulation
enhances the induction of chemokines in cumulus cells, we examined the effect of indomethacin (an inhibitor of PG biosynthesis) on
chemokine induction. Indomethacin was administered to mice atthe time of hCG injection, COCs were isolated at 14 h, andchemokine expression was examined by real-time RT-PCR and
immunofluorescence analysis. Significant increases in the level of
Ccl expression and higher fluorescence signal intensities for CCL2
were detected in the cumuli isolated from indomethacin-treated
Tamba et al.

WT mice than those from control animals (Fig. 1B and data notshown), suggesting that PG production in vivo is linked to the
suppression of chemokine expression in cumuli.
We then examined whether exogenously added PGE2 can suppress Ccl gene expression in the cumulus cells. WT and Ptger2⫺/⫺
COCs were collected from oviducts and incubated in vitro in media
containing 1 ␮M PGE2, 2 mM dbcAMP (a membrane-permeablecAMP analog), or 10 ␮M indomethacin for 3 h. The mRNA levelsof the chemokines were subsequently quantified by real time
RT-PCR (Fig. 1D). PGE2 and dbcAMP suppressed the expressionof Ccl9 in WT COCs, whereas indomethacin augmented theexpression by twofold, suggesting that the endogenous PGE2cAMP system participates in the negative regulation of Ccl9
expression. T⫺/⫺he basal expression level of Ccl9 mRNA was about
fourfold higher in Ptger2⫺/⫺ COCs than in WT COCs. Whereas
dbcAMP decreased the expression level of Ccl9, both PGE2 andindomethacin failed to alter the expression levels, indicating that the
suppression of Ccl9 expression by PGE2 is mediated through theEP2 receptor. Similar results were obtained in the gene expression
analysis for Ccl2 (Fig. 1D, lower panels) and Ccl7 (data not shown).
These findings indicate that Ccl expression is induced in the cumuli
after ovulation, but its expression level is negatively regulated by the
autocrine action of PGE2 through the EP2-cAMP system.

Ptger2⫺/⫺ COCs demonstrated a lower efficiency of in vitro fertilization (IVF) compared with WT COCs. Considering the enhanced
chemokine production in the Ptger2⫺/⫺ COCs, we next exploredwhether the addition of C-C chemokines would affect the IVF
processes. When we isolated WT COCs and incubated them with
sperm for 8 h, 75–80% of the oocytes were fertilized. We next added
100 ng/ml of CCL2, CCL9, or CCL7 to the medium containing
COCs 30 min before sperm addition and examined the effect ofthese chemokines on the fertilization rates. CCL9 and CCL7 areligands for CCR1, and CCL2 and CCL7 are ligands for CCR2 (20).
The addition of CCL2 and CCL9 decreased the fertilization rate
significantly, to 48.4 ⫾ 3.2% and 46.3 ⫾ 2.5%, respectively. The
addition of CCL7 decreased the fertilization rate further to 31.1 ⫾
6.0%; this inhibitory effect of CCL7 was abolished by the additionof an antagonist for CCR1/CCR2, vMIP-II (21) (Fig. 2A). In
contrast, CCL22, a ligand for CCR4, which was not detected in the
cumuli by RT-PCR analysis, did not affect the fertilization rate. In
addition, CCL2, CCL7, and CCL9 did not affect the fertilization
rates of cumulus-free oocytes. These findings demonstrate that
CCR1/CCR2 signaling suppresses fertilization by acting on cumulus
cells and raise the hypothesis that the impaired fertilization in the
Ptger2⫺/⫺ mice is due to the enhanced actions of these chemokines.
To test this hypothesis, we added vMIP-II to the medium containing
WT and Ptger2⫺/⫺ COCs 30 min before sperm addition andexamined the effect of the antagonist on the fertilization rate of
Ptger2⫺/⫺ COCs. vMIP-II significantly enhanced the fertilization
rate of Ptger2⫺/⫺ COCs but had no effect on the fertilization rate of
WT COCs (Fig. 2B).
We also examined the effect of in vivo indomethacin administration on fertilization and the role of CCR1/CCR2 signaling in thefertilization rate of indomethacin-treated COCs. Indomethacin
administration at the time of hCG injection significantly decreased
the fertilization rate both in vivo and in vitro (in vivo: control, 87.5%⫾ 1.7% vs. indomethacin, 46.7% ⫾ 4.4%; in vitro: control, 79.7%⫾ 1.7% vs. indomethacin, 45.0% ⫾ 2.1%). The addition of vMIP-II
partially but significantly improved the IVF rate of indomethacintreated COCs (Fig. 2B). These findings suggest that the impaired
fertilization in Ptger2⫺/⫺ and indomethacin-treated mice is due, atleast in part, to the increased production of chemokines.
Although Ptger2 deficiency enhances the production of chemokines by cumuli, these chemokines are produced at significant
concentrations by WT cumuli with intact EP2 signaling (Fig. 1 A).
Tamba et al.

Fig. 2. Chemokines facilitated sperm migration to cumuli, but their excessive
signaling on cumuli suppressed fertilization. (A) CCL2, CCL7, and CCL9, but not
CCL22, suppressed fertilization of WT COCs (cumulus-enclosed). The inhibition by
CCL7 was abolished by addition of the CCR1/CCR2 antagonist, vMIP-II (vMIP). Suchinhibitory effects were not observed in the absence of cumulus cells (cumulusfree). *, P ⬍ 0.01 (vs. control); †, P ⬍ 0.01 (vs. CCL7 only). (B) Restoration of thefertilization rate by vMIP-II in EP2 signal– depleted COCs. vMIP-II (vMIP) itself did
not alter the fertilization rate of control COCs (WT, Cont), but significantly
restored the rates of COCs from Ptger2⫺/⫺ (KO) or indomethacin-treated mice
(Indo). Values are presented as mean ⫾ SEM (18 –25 eggs for each well; n ⫽ 6). *,P ⬍ 0.01 (vs. control of WT or vehicle-treated mice); †, P ⬍ 0.01 (vs. without
vMIP-II). (C) Effects of CCL7 and vMIP-II on ovulation and fertilization in vivo. WT
female mice were injected i.p. with vehicle (-), CCL7 (50 ␮g/kg), or vMIP-II (250
␮g/kg) at 8 h after hCG injection and then mated with males overnight. Values arepresented as mean ⫾ SEM (n ⫽ 6 –7). *, P ⬍ 0.05 (vs. vehicle). (D) CCL7 and COC
facilitated sperm migration in a vMIP-II-sensitive manner. Sperm were subjected
to a Boyden chamber assay. The upper or lower wells were filled with medium
only (⫺) or medium containing CCL7 (100 ng/ml). Sperm (1 ⫻ 105 cells/ml) wereadded into the upper wells; 1 h later, the number of sperm that migrated into thelower wells was counted. Alternatively, the lower wells were filled with medium
only (-), medium containing CCL7, or WT or Ptger2⫺/⫺ (KO) COCs, and sperm wereadded into the upper wells in the presence or absence of vMIP-II (1 ␮g/ml). Values
are presented as mean ⫾ SEM (18 –24 COCs for each well; n ⫽ 4). *, P ⬍ 0.001 (vs.
control); †, P ⬍ 0.001 (vs. without vMIP-II). Representative results of two (A–C) or
three (D) independent experiments are shown.

Consequently, we next addressed the physiological significance ofchemokine signaling in the cumulus. CCL7 or vMIP-II was administered intraperitoneally (i.p.) to WT mice at 8 h after hCGadministration (just before ovulation), and their effects on ovulation and fertilization were examined in vivo. Although neither
compound significantly altered the ovulation number (Fig. 2C),
CCL7 decreased the fertilization rate, and, interestingly, vMIP-IIsignificantly affected in vivo fertilization, although this compound
exhibited no significant effects on the IVF rate of WT COCs. Thesefindings indicate that endogenous CCR1/CCR2 signaling not only
negatively regulates fertilization, but also is required for proper
fertilization. Given that CCR1 signaling has chemoattractant activity for sperm in vitro (6), we suspected that chemokine signaling
could facilitate fertilization through its chemoattractant activity for
sperm. To test this possibility, we used a Boyden chamber assay to
PNAS 兩 September 23, 2008 兩 vol. 105 兩 no. 38 兩 14541

MEDICAL SCIENCES

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Chemokines Facilitate Sperm Migration to Cumuli, but Their Excessive
Signaling on Cumuli Suppresses Fertilization. As reported previously,Downloaded by guest on November 4, 2021

14542 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0805699105

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ing on cumuli suppress fertilization? One difference between WT
and Ptger2⫺/⫺ COCs is in the viscosity of ECMs. During our IVF
experiments, we noted that Ptger2⫺/⫺ COCs were more sticky andmore resistant to hyaluronidase treatment compared with WT
COCs. Hyaluronan is a major component of the cumulus ECM, andproduction of hyaluronan induces cumulus expansion (22). Spermuse their own hyaluronidase to penetrate the cumulus ECM layer.
If excessive chemokine signaling indeed induced the resistance of
cumulus ECM to hyaluronidase, this could interfere with sperm
penetration. Consequently, we examined the hyaluronidase sensitivity of Ptger2⫺/⫺ COCs. The COCs were exposed to 0.01%
hyaluronidase for 15 min, and the number of nondenuded eggs wascounted. Both the WT and Ptger2⫺/⫺ COCs responded to hyaluronidase treatment, but the Ptger2⫺/⫺ COCs were significantly
more resistant to hyaluronidase than the WT COCs; 38% of thePtger2⫺/⫺ COCs retained several layers of cumulus cells, whereas
only 10% of the WT COCs were nondenuded under this condition(Fig. 3A). We next evaluated the total hyaluronan content in WT
and Ptger2⫺/⫺ COCs. No significant difference in hyaluronan
content was found between the WT and Ptger2⫺/⫺ COCs (data notshown). Based on these findings, we hypothesized that the organization of ECM components other than hyaluronan is altered in
Ptger2⫺/⫺ COCs and may cause the retention of cumulus cells
around the oocyte.
Key molecules in the regulation of cell-to-ECM organization are
integrins, which bind ECM components, such as fibronectin andlaminin, in a divalent cation-dependent manner (23). To investigate
the possible involvement of integrins in the cumulus cell adhesion,
we examined the effect of EDTA on ECM resistance to hyaluronidase in Ptger2⫺/⫺ COCs. We found that although EDTA itself did
not induce the dissociation of cumulus cells in the absence of
hyaluronidase, the addition of EDTA resulted in the complete
disappearance of nondenuded COCs in the Ptger2⫺/⫺ group (Fig.
3A), suggesting that molecules mediating divalent cation-dependentadhesion, such as integrins, are involved in the resistance tohyaluronidase in Ptger2⫺/⫺ COCs. Resistance to hyaluronidase also
was found in CCL7-treated WT COCs, and this effect of CCL7 wasblocked by vMIP-II. The effect of CCL7 also was abolished by an
integrin-inhibitor peptide, GRGDS, but not by a control peptide(Fig. 3B). These findings suggest that the integrins and their binding
to RGD-containing ECM proteins are involved in ECM resistanceto hyaluronidase found in the Ptger2⫺/⫺ COCs and in WT COCs
treated with CCL7. Indeed, when we stained the Ptger2⫺/⫺ COCs
with an anti-integrin ␤1 antibody in a nonpermeabilized condition,
we detected strong signals on the surface of the cumulus cells. We
failed to detect such strong signals on the surface of cumulus cellsof WT COCs, however (Fig. 3C). We found similar results for thesurface accumulation of integrin ␣v in the cumulus cells (Fig. S1).
We detected no significant changes in the gene expression levels of
integrin ␤1 (Itgb1) or ␣v (Itgav) between WT and Ptger2⫺/⫺ cumuli(data not shown). We also found that in vitro CCL7 treatment
stimulated the accumulation of integrins ␤1 and ␣v on the cumulus
cell surface (Fig. 3C and data not shown). These findings demonstrate that the loss of EP2 signaling, and an input of chemokine
signaling, in cumulus cells led to integrin clustering on the cellsurface. Moreover, fibronectin densely accumulated in the ECM of

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Failure in Ptger2ⴚ/ⴚ COCs. Why does excessive CCR1/CCR2 signal-

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evaluate whether ovulated COCs facilitate sperm migration toward
COCs themselves. We used CCL7 as a control and found that CCL7
significantly stimulated sperm chemotaxis without affecting sperm
chemokinesis, and that both WT and Ptger2⫺/⫺ COCs facilitatedsperm migration toward COCs under the same conditions (Fig.
2D). Moreover, the stimulating effects of COCs were suppressed by
the addition of vMIP-II. These finding suggest that endogenous
CCR1/CCR2 signaling facilitates in vivo fertilization by stimulating
sperm migration toward cumuli.

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Fig. 3. Ptger2 deficiency and CCL7 enhanced cumulus ECM assembly in an
integrin-dependent manner. (A) The ECM of Ptger2⫺/⫺ COCs showed divalentcation-dependent hyaluronidase resistance. WT and Ptger2⫺/⫺ COCs (KO) wereincubated with 0.01% hyaluronidase in the presence or absence of 1 mM EDTA
for 15 min. The number of oocytes surrounded by the cumulus layer was counted.
A typical view of an oocyte for each treatment is shown. Arrowhead, egg surface.
*, P ⬍ .01 (vs. WT). (B) CCL7-induced hyaluronidase resistance in the cumulus ECM.
COCs were incubated with CCL7 in combination with vMIP-II (vMIP), EDTA, anintegrin inhibitor (RGD), or a control peptide (DGR) for 3 h and then subjected totreatment with 0.01% hyaluronidase. Values are presented as mean ⫾ SEM(20 –26 COCs for each well; n ⫽ 4). *, P ⬍ .01 (vs. Cont); †, P ⬍ .01 (vs. CCL7 only);#P ⬍ .05. (C) Surface accumulation of integrin ␤1 in the Ptger2⫺/⫺ and CCL7treated cumulus cells. WT, Ptger2⫺/⫺ COCs (KO), or WT COCs treated with CCL7
were stained with a fluorescein isothiocyanate (FITC)-labeled anti-mouse integrin
␤1 antibody in a nonpermeabilized condition. (D) Enhanced accumulation offibronectin in the Ptger2⫺/⫺ cumulus ECM. (E) Ptger2 deficiency resulted inincomplete cumulus ECM disassembly and sperm capture by cumuli during IVF. In
the WT COCs, individual cumulus cells were scattered, and sperm passed between
the cells (arrowhead). In the Ptger2⫺/⫺ COCs (KO), cumulus cells remained inclusters, and some sperm (arrows) were captured by cumulus cells. CCL7 treatment mimicked the effect of Ptger2 deficiency, but an integrin inhibitor (RGD)
suppressed the effect of CCL7. (Scale bars: A, C, and D, 100 ␮m; E, 20 ␮m.)
Representative results of three (A and B) or five (C–E) independent experiments
are shown.

Ptger2⫺/⫺ COCs, whereas low levels of fibronectin were detected in
the ECM of WT COCs (Fig. 3D). Based on these results, weconclude that excessive chemokine signaling in the cumulus cells
facilitates the surface accumulation of integrins, leading to theexcessive formation and assembly of fibronectin fibrils and conferring the cumulus ECM with resistance to hyaluronidase in Ptger2⫺/⫺
COCs.
To evaluate whether this integrin-mediated resistance of the
ECM to hyaluronidase is sufficient to affect the disassembly of the
cumulus ECM by sperm, we examined the appearance of cumuli
after incubation of WT and Ptger2⫺/⫺ COCs with sperm for 1 h. In
the WT COCs, sperm disassembled the cumulus ECM, and theTamba et al.

Discussion
Integrin–Fibronectin Assembly in the Maintenance of ECM Status.

Accumulating evidence has suggested that the orchestrated organization of the oocyte, cumulus, and ECM is essential for successful
fertilization. In the cumulus ECM, hyaluronan is known to be themajor component responsible for the viscoelastic properties andexpansion of COCs. Recent studies have shown that several proteins, including inter-␣-trypsin inhibitor, act as ‘‘nodes’’ to cross-link
multiple hyaluronan chains, and that this hyaluronan network isessential for in vivo fertilization (11–13). In the present study, we
have demonstrated that the chemokine/PGE2 system regulates theassembly of a different cumulus ECM component, an integrinmediated fibronectin assembly, and that this pathway plays animportant role in the fertilization process. Fibronectin is a polymorphic adhesive glycoprotein present in biological fluids (solubleform) and the ECM (insoluble form), with its fibril formation
controlled by its binding to integrins on neighboring cells (24).
Fibronectin is produced by cumulus cells located in the innermost
layer, the corona radiata cells, which functionally interact with theoocyte even after ovulation (25, 26). Indeed, fibronectin is present
most abundantly around the innermost layer in the WT cumulus
ECM (Fig. 3D), and the cumulus ECM, showing resistance tohyaluronidase, is the innermost layer in Ptger2⫺/⫺ COCs (Fig. 3A).
These properties indicate that fibronectin assembly is dynamic andpresumably regulated by chemokine production. Once excessive
fibronectin fibrils are formed by chronic exposure of cumulus cells
to chemokines, these fibrils may serve as a barrier, providing
resistance to hyaluronidase and disturbing sperm penetration
through the cumulus ECM layer. The balance between chemokines
and PGE2 controls the cumulus surface expression of integrins,
maintaining an ECM status suitable for fertilization.

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Capture of Sperm by the Cumulus. Capture of sperm by the cumulus

cells may be another mechanism underlying impaired fertilizationin the Ptger2⫺/⫺ COCs. Integrin ␣6␤1 on the oocyte has beensuspected to be a potential receptor for fertilin ␤ and cyritestin, twomembers of the ADAM family of proteins present on the sperm
surface (27). The current study demonstrates that accumulation of
Tamba et al.

A

B

Fig. 4. Schematic model of the interactions of chemokines and PGE2 signaling in cumulus ECM disassembly and successful fertilization. (A) Ovulationassociated signals induced gene expression and production of chemokines,
such as CCL7, in cumulus cells. CCL7 stimulated cumulus ECM protein assembly
to protect the oocyte in an autocrine manner and facilitated sperm migration
to COCs. (B) In cumulus cells, CCL7 stimulated the surface accumulation of
integrins, thereby enhancing fibronectin fibril formation (left). Once the COCs
reached the fertilization site, PGE2/EP2/cAMP signaling down-regulated theseactions of chemokines on the cumuli (right); however, in Ptger2⫺/⫺ cumuli(left), chemokine signaling persisted and interfered with sperm penetration
because of its hyaluronidase resistance and the direct binding between integrins (cumulus) and fertilin (sperm).

integrins ␤1 and ␣v is augmented on the surface of EP2 signal–
depleted cumulus cells (Fig. S1), that some motile sperm are boundto a cluster of the cumulus cells (Fig. 3E). Moreover, CCL7 induces
sperm capture, which is reversed by an integrin inhibitor peptide
and by EDTA (Fig. 3E and Table S2), indicating that capture of
sperm by the cumulus also is due to augmented surface accumulation of integrins. Thus, sperm may be captured by cumulus cells
through the binding of integrins (cumulus) and ADAM proteins(sperm). Indeed, an anti-ADAM2 antibody was found to effectively
suppress sperm capture without affecting cumulus clusters (datanot shown).
Stimulants for Ccl Gene Expression. Which signals initiate chemokine

gene expression after ovulation? The COCs are exposed to widely
varying circumstances after ovulation. Ovulation and follicularrupture are characterized by inflammation-like processes, such as
matrix metalloproteinase expression and cytokine production (28).
Gene expression of interleukin-1␤ (IL-1␤; Il1b) in the ovary has
been shown to increase after ovulation (29). This cytokine is known
to stimulate CCL2 production in various cell types (30, 31). Recently, it was reported that IL-1␤ stimulates CCL2 production inhuman granulosa cell culture (32). Thus, IL-1␤ is a strong candidate
molecule for the stimulation of Ccl gene expression in the cumulus.
In this respect, it is interesting that the expression Il1b was increasedin Ptger2⫺/⫺ cumuli (Table S1), and thus synergistic interactions in
the gene expression of Ccl and Il1b may exist in the cumulus cells.
PNAS 兩 September 23, 2008 兩 vol. 105 兩 no. 38 兩 14543

MEDICAL SCIENCES

cumulus cells were stripped from the eggs and individually scatteredapart from one another. The sperm often came in contact with thedispersed cumulus cells but were able to pass between the cumulus
cells (Fig. 3E). In contrast, in the Ptger2⫺/⫺ COCs, most of the
cumulus cells were incompletely dispersed, still forming clusters,
indicating resistance of the cumulus ECM to sperm hyaluronidase.
In addition, the cumulus cells captured some motile sperm, with theheads of the sperm adhering to the cumulus surface. This adhesion
appeared to be tight and irreversible, because captured sperm were
never released (Fig. 3E). Prominent surface expression of integrin
␤1 and ␣v was still detected on the cell surface of such cumulus
masses (data not shown). Both cumulus cluster formation (i.e.,
ECM resistance to hyaluronidase) and sperm capture were observed in a similar analysis of indomethacin-pretreated WT COCs
and CCL7-treated WT COCs (Fig. 3E, Movies S1 and S2), in whichsurface accumulation of integrin was enhanced. An integrininhibitor peptide, and EDTA, reversed CCL7-induced cumulus
cluster formation and sperm capture (Fig. 3E and data not shown),suggesting that integrins are involved not only in ECM resistance tohyaluronidase, but also in sperm capture.
Taken together, our findings highlight the two distinct roles ofchemokines in fertilization; chemokine signaling strengthens theassembly of the cumulus ECM during transition into the ampulla
facilitates sperm migration toward COCs. Our findings further
demonstrate that if these chemokine actions persist without downregulation, they interfere with the disintegration of the cumulus
ECM by sperm, and that PGE2-EP2 signaling suppresses thischemokine signaling and facilitates disintegration of the cumulus
ECM by sperm for successful fertilization (Fig. 4).

Moreover, because IL-1␤ also has been shown to induce theexpression of Ptgs2 and Ptges (PGE synthase gene) in various cells(33, 34), PGE2 synthesis in cumulus cells may be resumed by theaction of IL-1␤. If this mechanism were operating, then IL-1␤ could
determine the timely interaction between PGE2 and CCL actionsin the cumulus ECM assembly.
In summary, we have demonstrated that chemokine signaling
facilitates both sperm attraction to the COC and COC compaction
by the integrin-mediated cumulus ECM assembly, that PGE2-EP2
signaling acts as a negative regulator of chemokine action tosensitize the cumulus ECM to disassembly by sperm hyaluronidase.
These findings contribute to our understanding of not only themechanism of the promotion of fertilization by PGE2, but also theimportance of chemokine signaling in the regulation of the cumulus
ECM, and sperm guidance, for successful fertilization.
Materials and Methods
Detailed methods are provided in the SI Text. An abbreviated summary isprovided here.

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Animals and Hormone Treatment. Follicular growth and ovulation were stimulated in WT and Ptger2⫺/⫺ mice at 3 weeks of age using the following
hormonal regimen: 5 IU of pregnant mare serum gonadotropin (PMSG) was
injected i.p., followed 48 h later by 5 IU of hCG injected i.p. Alternatively, WT
female mice were injected subcutaneously (s.c.) with indomethacin (5 mg/kg)
simultaneously with the hCG injection.

animals (Fig. 1 A), or from each animal (Fig. 1B) and COC RNA (Fig. 1D), wassubjected to real-time RT-PCR. Primer sequences and PCR conditions are given in
Table S3. The specificity of the primers was confirmed by checking the productsize through gel electrophoresis and the melting temperature. Oviductal sections
containing COCs were fixed and incubated with an antibody specific for CCL2. For
integrin ␤1 detection, isolated COCs were incubated with FITC-conjugated hamster anti-rat CD29 antibody in detergent-free conditions.
IVF and Hyaluronidase Resistance. IVF was performed as described in ref. 19.
Briefly, COCs were incubated with sperm in IVF medium supplemented with aphysiological concentration of chemokines (100 ng/ml; 7.7 nM each) or vMIP-II(1 ␮g/ml). After 12 h, fertilized eggs were identified by the formation ofpronuclei. COCs were incubated in 0.01% hyaluronidase for 15 min, and thenumber of oocytes with several cumulus layers was counted. To examine theresistance of the ECM to sperm-derived hyaluronidase, COCs were incubatedwith sperm for 1 h, and stripped cumulus cells were examined.
Statistical Analyses. Data are expressed as mean ⫾ SEM. Comparison of twogroups was analyzed using the Student t test. For comparison of more than
two groups with comparable variances, one-way ANOVA was performed first,
then Tukey’s test was used to evaluate the pairwise group difference.

Real-Time RT-PCR and Immmunofluorescence. Cumulus cells were collected from
WT or Ptger2⫺/⫺ mice at 3, 9, and 14 h after hCG injection. The COCs wereincubated with vehicle, PGE2, dbcAMP, or indomethacin. Cumulus RNA from five

ACKNOWLEDGMENTS. We thank Drs. Y. Noda and M. Hirose (Department of
Obstetrics and Gynecology, Shiga University of Medical Science) for theirinvaluable advice and technical support; Dr. K. Nakayama for precious advice
and continuous support; and Dr. H. A. Popiel and Ms. Y. Nakaminami forcareful reading of the manuscript and secretarial assistance, respectively. This
work was supported in part by Grants-in-Aid for Scientific Research and on
Priority Areas Applied Genomics and Mechanisms of Sex Differentiation from
the Ministry of Education, Culture, Sports, Science, and Technology of Japan,
from the Ministry of Health and Labor of Japan, and the National Institute of
Biomedical Innovation of Japan. S.T. is a recipient of a Research Fellowshipfrom the Japan Society for the Promotion of Science for Young Scientists.

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14544 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0805699105

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