1 Department of Public Health and Cell Biology, University of Rome Tor Vergata,
Rome 00133, Italy
2 Department of Immunology and Cell Biology, Mario Negri Institute for
Pharmacological Research, Milan 20157, Italy
3 Department of Genetics, Biology and Biochemistry, University of Turin, Turin
10126, Italy
4 Institute of Endocrinology, Università degli Studi di Milano, Milan
20100, Italy
5 SigmaTau SpA, Pomezia, Rome 00040, Italy
6 MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford,
Oxford OX1 3QU, UK
7 Microbiology Section, Department of Experimental Medicine and Biochemical
Sciences, University of Perugia, Perugia 05122, Italy
8 Centro IDET, Institute of General Pathology, Università degli Studi di
Milano, Milan 20100, Italy
Author for correspondence (e-mail:
mantovani{at}marionegri.it)
Accepted 22 December 2003
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SUMMARY |
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Key words: Cumulus oophorus, Extracellular matrix, Fertility, Pentraxins, Mouse
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Introduction |
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This matrix is synthesized by cumulus cells a few hours before ovulation
and, in the mouse, the combined action of gonadotropins and soluble oocyte
factors is required for triggering this process
(Buccione et al., 1990;
Salustri et al., 1990b
). The
major component is hyaluronan (HA), a large polyanionic glycosaminoglycan
responsible for viscoelastic properties and expansion of the cumulus oophorus
(Salustri et al., 1992
). In
vitro and in vivo studies have shown that serum-derived inter-
-trypsin
inhibitor (I
I or ITI) is essential for HA-matrix assembly and mouse
female fertility (Chen et al.,
1992
; Chen et al.,
1996
; Zhuo et al.,
2001
; Fulop et al.,
2003
). I
I consists of a small protein, named bikunin or
light chain, with a chondroitin sulfate moiety that connects two additional
proteins, named heavy chains. The heavy chains of I
I are covalently
transferred to HA in the preovulatory follicle. It has been proposed that
these polypeptides stabilize the HA-matrix by cross-linking HA molecules
through covalent and ionic bonds. Another crucial component of the cumulus
matrix is tumour necrosis factor
-induced protein 6 (TNFAIP6 or TSG6),
a multifunctional protein usually associated with inflammation, which has the
ability to specifically bind HA (Lee et
al., 1992
; Milner and Day,
2003
). TNFAIP6 is synthesized by cumulus and granulosa cells in
the preovulatory follicle (Fulop et al.,
1997
; Yoshioka et al.,
2000
), and Tnfaip6-deficient mice are unable to form
stable cumulus matrix and are sterile
(Fulop et al., 2003
). Covalent
transfer of heavy chains from I
I to HA does not occur in
Tnfaip6/ mice, indicating that TNFAIP6 is a
key catalyst in this reaction. Oocytes, besides promoting cumulus matrix
synthesis, inhibit hormone-induced proteolytic enzyme expression by mouse
cumulus cells during matrix deposition, probably providing an additional
mechanism for matrix stabilization
(Canipari et al., 1995
).
Indeed, synthesis of cumulus matrix ceases at ovulation and its degradation
begins a few hours later, coinciding with an increase in protease production
by the oocyte and the cumulus cells
(D'Alessandris et al.,
2001
).
Recent findings have shown that the long pentraxin 3 (PTX3) is also
involved in cumulus matrix stability
(Varani et al., 2002).
Pentraxins are a superfamily of conserved proteins, characterized by a cyclic
multimeric structure (Emsley et al.,
1994
). The classical short pentraxins, C-reactive protein and
serum amyloid P component, are acute phase proteins produced in the liver in
response to inflammatory mediators (Steel
and Whitehead, 1994
; Szalai et
al., 1997
). Long pentraxins have an unrelated long N-terminal
domain coupled to the C-terminal pentraxin domain, and differ in their gene
organization, chromosomal localization, cellular source and inducing stimuli,
as well as in the ligands they recognize
(Mantovani et al., 2003
).
PTX3, the first long pentraxin identified
(Breviario et al., 1992
;
Lee et al., 1993
), is produced
as a 10-20 subunit multimer protein by macrophages and other cell types or
tissues upon stimulation with primary inflammatory mediators
[lipopolysaccharide, interleukin 1 (IL1), tumor necrosis factor
(TNF
)] (Breviario et al.,
1992
; Lee et al.,
1993
; Lee et al.,
1994
; Introna et al.,
1996
; Bottazzi et al.,
1997
). PTX3 appears to have a protective effect in inflammatory
sites limiting tissue damage, possibly by regulating apoptotic cell clearance
(Mantovani et al., 2003
;
Ravizza et al., 2001
). It also
binds to selected microorganisms facilitating their recognition by macrophages
(Garlanda et al., 2002
). As a
consequence, Ptx3 deficiency renders mice susceptible to selected
pathogens. Furthermore, Ptx3/ mice show a
severe defect in female fertility
(Garlanda et al., 2002
;
Varani et al., 2002
). It has
been reported that Ptx3 is expressed by cumulus cells before
ovulation, and that infertility of Ptx3 deficient mice is due to
defects in ovulation and oocyte fertilization, associated with loss of cumulus
investment during extrusion from the ovary
(Varani et al., 2002
). The
molecular basis for the loss of cumulus integrity in Ptx3 deficient
mice is currently unknown. It has been hypothesized that PTX3 might have
antiproteolytic activity and functions to protect the oocyte and the
extracellular matrix from proteases involved in rupture of the follicle
wall.
We show that oocytes ovulated by Ptx3/
mice can be fertilized in vitro, indicating that the oocyte develops normally
in the absence of PTX3, and that a defective cumulus expansion is the major
cause for in vivo fertilization failure. PTX3 is produced by cumulus cells
both in vivo and in vitro under stimuli inducing cumulus expansion, and
localizes in the extracellular matrix. We also show that presence of PTX3 is
essential for the expanding cumulus to retain HA molecules in the
intercellular spaces, although it cannot prevent nor delay matrix degradation,
which occurs at later times. The PTX3 role in retaining HA is independent of
II incorporation into the matrix, but it is likely to be mediated by
TNFAIP6. Finally, we show that PTX3 is expressed by human cumulus
cells as well, and that PTX3 protein is present in human cumulus matrix,
suggesting that this molecule might have the same role in human female
fertility.
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Materials and methods |
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Procedures involving animals and their care conformed with institutional guidelines in compliance with national (4D.L. N.116, G.U., Suppl. 40, 18-2-1992) and international law and policies (EEC Council Directive 86/609, OJ L 358,1,12-12-1987; NIH Guide for the Care and Use of Laboratory Animals, US National Research Council 1996). All efforts were made to minimize the number of animals used and their suffering.
In vivo and in vitro fertilization
Cumuli oophori, zygotes and embryos were recovered from the oviducts of
untreated females after natural mating or after hormonally-induced
superovulation (Hogan et al.,
1994). Cumulus oophorus matrix was digested with
Streptomyces hyaluronidase (Calbiochem), and cumulus cells and
oocytes were separated as described (Hogan
et al., 1994
).
In vitro fertilization (IVF) was performed using intact oocytes
(Hogan et al., 1994) or zona
pellucida free eggs stained with 1 µg/ml of Hoechst 33258 (Sigma-Aldrich)
(Conover and Gwatkin, 1988
).
After insemination embryos were cultured in KSOM media (Cell & Molecular
Technologies). Sperm-egg fusion was determined by counting eggs with
fluorescent fertilizing sperm 4 hours after insemination of zona free eggs.
Fertilization was assessed by counting two-cell stage embryos and blastocysts,
1 or 4 days after insemination of intact oocytes, respectively. Embryo
transfer was performed as described (Hogan
et al., 1994
).
PTX3 mRNA and protein
Northern blot analysis, in situ hybridation, expression and purification of
PTX3, and antibody assays were performed as described
(Biffo and Tolosano, 1992;
Introna et al., 1996
;
Bottazzi et al., 1997
). Human
PTX3 was expressed in CHO cells and purified under endotoxin-free conditions
by immunoaffinity with a rat mAb (MNB4)
(Muller et al., 2001
).
Purified PTX3 was checked for purity by SDS-PAGE, and for lipopolysaccharide
(LPS) contamination by Limulus amebocyte lysate assay
(Bio-Whittaker).
Isolation and culture of cumuli
Ovaries were dissected from 8- to 12-week-old mice injected 48 hours
earlier with 5 IU pregnant mares' serum gondotropin (PMSG), and cumulus
cell-oocyte complexes (COCs) were mechanically isolated. COCs were cultured in
drop, under mineral oil, of MEM supplemented with 1% fetal bovine serum (FBS),
3 mM glutamine, 0.3 mM sodium pyruvate and 50 ng/ml gentamycin, in the
presence of 100 ng/ml FSH (highly purified rat-FSH; kindly provided by the
NIDDF and the National Hormone and Pituitary Program, NIH, MD, USA), or 1 mM
8-Bromo cyclic cAMP (8Br cAMP; Sigma), or 1 ng/ml epidermal growth factor
(EGF; Sigma), or 200 ng/ml prostaglandin E2 (PGE2;
Sigma), at 37°C, 5% CO2, for the time indicated in the text. In
certain cases, human recombinant PTX3 was added at the beginning of culture.
Cultures of isolated cumulus cells were generated by mechanical dissociation
of the COCs in the culture drop and removal of the oocytes, as described
(Salustri et al., 1990b).
Ovulated COCs were collected from the oviducts 14 hours after the injection of 5 IU human chorionic gonadotropin (hCG) into PMSG-primed mice, and cultured in MEM, supplemented as reported above, at 37°C, 5% CO2 for the time indicated in the text.
Human cumulus cells and cumulus matrix were obtained from patients undergoing IVF.
Western analysis
Mouse COCs and human cumulus fragments were directly solubilized in a
reducing Laemmli loading buffer, or treated with 1 U Streptomyces
hyaluronidase (Calbiochem) for 2 hours at 37°C in the presence of protease
inhibitors (Boehringer Mannheim) before adding loading buffer. In certain
cases, the culture medium was collected at the end of culture and mixed with
an equal volume of 2xloading buffer. For PTX3 localization analysis,
after digestion of COCs with Streptomyces hyaluronidase, oocytes were
collected under the microscope and the sample microcentrifuged. Oocytes, cell
pellet and supernatant (matrix extract) were mixed with loading buffer.
Protein extracts were separated by SDS-PAGE (7% acrylamide).
Western-blotting analysis of PTX3 was performed by using a polyclonal antibody
against mouse PTX3 (1 µg/ml) and monoclonal antibody 16B5 against human
PTX3 (1 µg/ml), for mouse and human cumulus extracts, respectively. For
II immunoblotting, a polyclonal antibody against human I
I
(1:2000; Dako) was used.
Quantitation of HA
Compact COCs were stimulated with 100 ng/ml FSH, 1% fetal bovine serum
(FBS), in the presence of [35S]-sulfate (60 µCi/ml) and
[3H]-glucosamine (100 µCi/ml; NEN Life Science Products,
Zaveten, Belgium), for the time indicated in the text, at 37°C in 5%
CO2. Medium and cell-matrix were collected separately, and the
amount of HA in the two compartments determined as described elsewhere
(Camaioni et al., 1993).
Immunofluorescence analysis of PTX3 and HA
For localization studies of PTX3 and HA, COCs were incubated with 10
µg/ml rabbit anti-mouse PTX3 polyclonal antibody and 5 µg/ml
biotinylated HA binding protein (HABP; Seikagaku) in phosphate buffer with 3%
BSA for 2 hours at room temperature. After washing, COCs were incubated with
FITC-labelled anti-rabbit IgG and streptavidin AlexaFloor 568 (Molecular
Probes) for 1 hour in phosphate buffer with 3% BSA at room temperature. Nuclei
were stained with Hoechst 33258. Cumuli were visualized with a fluorescence
microscope.
PTX3 binding to hyaluronan and TNFAIP6
The interaction of PTX3 with recombinant human TNFAIP6
(Nentwich et al., 2002), or
with the Link module from human TNFAIP6 (termed Link_TNFAIP6)
(Kohda et al., 1996
), was
investigated using colorimetric microtitre plate assays essentially as
described before (Mahoney et al.,
2001
). Initial experiments (in PBS, 0.05% Tween-20, at room
temperature) compared the binding of biotinylated-PTX3 (bPTX3; 1000 ng/well,
which corresponds to 22.2 pmol/well assuming a molecular mass of 45 kDa for
the PTX3 protomer) and biotinylated-HA (12.5 ng/well) to plates coated with 25
pmol/well full-length TNFAIP6. All other assays were carried out in 50 mM
Na-acetate, 100 mM NaCl, 0.05% Tween-20 (pH 6.0) [conditions that are optimal
for the interaction of HA with Link_TNFAIP6
(Parkar et al., 1998
)], and
measured the binding of bPTX3 (5-1000 ng/well) to Link_TNFAIP6 coated wells
(25 pmol/well). Competition experiments were performed using 200 ng/well bPTX3
binding (i.e. a saturating amount) in the absence and presence of HA (0.1-2500
ng/well), Link_TNFAIP6 (2-5000 ng/well) or unlabelled PTX3 (2-5000 ng/well).
All absorbance measurements (405 nm) were corrected by subtracting values from
uncoated control wells.
PTX3 binding to sperm
Spermatozoa were isolated from the cauda epididymis and vas deferens of
male mice of proven fertility and capacitated for 1 hour at 37°C
(Hogan et al., 1994). Binding
of soluble PTX3 to spermatozoa was characterized by cytofluorimeter analysis,
and by immunofluorescence using 100 µg/ml FITC-labelled PTX3 and 10
µg/ml biotin-labelled PTX3, respectively.
For the adhesion assay, 35 mm cell culture plates were first coated with polylysine and, second, one half of the surface was layered with purified PTX3 (20 µg/ml, overnight at 4°C). PTX3 solution was then removed taking care not to spill into the other half of the surface. Plates were washed with PBS, filled with 50 mg/ml BSA in PBS and incubated at 37°C for 1 hour. Plates were washed and, subsequently, 106 sperm, suspended in 2 ml Whittingham's medium, were added. After 4 hours of incubation at 37°C, non-adherent cells were gently washed two times with PBS and the number of cells adhering to the two different coatings were blindly counted in eight random fields at 20x magnification.
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Results |
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Collectively, these data provide evidence that abnormalities in the cumulus underlie in vivo fertilization failure and infertility of Ptx3/ females, and strongly suggest that additional developmental defects are unlikely.
We also examined whether Ptx3 deficiency could affect the implantation process as well. We observed normal pregnancy and delivery (data not shown) when Ptx3+/+ blastocysts were transferred to the uterus of Ptx3/ pseudopregnant females, suggesting that implantation and subsequent processes are not altered in Ptx3/ mice.
PTX3 is produced by cumulus cells and localizes in the matrix
Ptx3 mRNA expression in naturally cycling mice was confined to
cumulus cells and to a few granulosa cells lining the follicle antrum of
preovulatory follicles, with no evidence of Ptx3 transcripts in
oocytes, peripheral granulosa cells, theca cells or interstitial ovarian
tissue (Fig. 2A). Northern blot
analysis of whole ovary revealed that Ptx3 mRNA expression starts 2
hours after injection of an ovulatory dose of hCG in PMSG-primed mice, peaks
at 6 hours and declines thereafter (Fig.
2B), showing close temporal correlation to matrix deposition by
cumulus cells and cumulus expansion
(Salustri et al., 1989;
Salustri et al., 1992
).
|
Previous studies have shown that cumuli oophori are induced to expand in
vitro by the combined action of a soluble factor produced by the oocytes and
follicle-stimulating hormone (FSH), or cyclic AMP, EGF or PGE2
(Salustri et al., 1990a;
Salustri et al., 1990b
;
Buccione et al., 1990
;
Tirone et al., 1997
). Results
reported in Fig. 3 show that
such synergistic action is also required to induce PTX3 expression by in vitro
cultured cumulus cells. FSH, 8Br-cAMP, EGF and PGE2 stimulated PTX3
synthesis by intact cumuli (Fig.
3A), but FSH, and all of the above-mentioned factors, failed to do
so when cumulus cells were dissected from the cumuli and cultured in the
absence of oocytes (Fig. 3B,
and data not shown). Interestingly, most PTX3 synthesized during in vitro
expansion was retained in the matrix, mimicking the in vivo preovulatory
condition.
|
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Double staining of Ptx3+/ ovulated cumuli for HA and PTX3 revealed that PTX3 co-localizes with HA in the extracellular matrix (Fig. 6). Indeed, PTX3 was organized in a fine fibril network, extending from the outer region of the cumulus to the zona pellucida, which appears to be intimately associated to HA.
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Discussion |
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Expression of PTX3 is induced by primary inflammatory signals in various
cell types in vitro and in vivo (Breviario
et al., 1992; Lee et al.,
1993
; Introna et al.,
1996
). Several lines of evidence, including the phenotype of COX2-
and prostaglandin receptor E receptor subtype EP(2)-deficient mice
(Lim et al., 1997
;
Davis et al., 1999
;
Hizaki et al., 1999
;
Tilley et al., 1999
), point to
analogies between the process of ovulation and inflammation
(Espey, 1994
;
Richards et al., 2002
). In
addition to a hormonal ovulatory stimulus, oocyte soluble factors are required
for eliciting HA synthesis and cumulus expansion
(Salustri et al., 1990b
;
Buccione et al., 1990
).
Likewise, we show here that expression of Ptx3 is induced in cumulus
cells by an ovulatory stimulus, and that the oocyte influences this response.
Ptx3 expression was also detected in granulosa cells lining the
antral cavity of the preovulatory follicle. This finding is consistent with
previous observations showing that such granulosa cell subpopulations
synthesize HA-rich matrix and become included in the expanded cumulus.
Experimental evidence suggests that a gradient of oocyte factor(s) is
established in the preovulatory follicle that influences cumulus cells as well
as antral granulosa cells (Salustri et
al., 1992
). Growth differentiation factor 9 (GDF9), is probably
the oocyte factor involved in the control of such processes
(Elvin et al., 1999
;
Varani et al., 2002
).
Therefore, interplay between different signals is likely to be required for
temporally and anatomically restricted PTX3 expression during the
periovulatory period. The synthesis of PTX3 during both ovulation and
inflammation adds a further element linking these processes.
PTX3 localizes in the cumulus matrix and plays a crucial role in cumulus
expansion. In Ptx3-deficient mice, corona radiata cells fail to
polarize and randomly surround the oocyte in the preovulatory follicle. After
ovulation, single COCs are no longer identifiable: the oocytes appear
scattered in an unstable, uniform mass that quickly disaggregates into single
cells. Experiments performed in vitro clearly show that
Ptx3/ cumuli are unable to retain HA within
the matrix. Presently, two additional molecules have been identified that are
involved in HA organization in the cumulus matrix, TNFAIP6 and II.
TNFAIP6 is a protein able to tightly bind HA through a link module
(Kohda et al., 1996
;
Milner and Day, 2003
). It is
produced by cumulus cells during the expansion process with a temporal pattern
identical to that of PTX3 and HA. I
I is a serum protein complex, formed
by two heavy chains and a light chain (bikunin) covalently linked to a
chondroitin sulfate moiety, which diffuses into the follicle after the
luteinizing hormone (LH) surge, when cumulus expansion is triggered
(Powers et al., 1995
).
Bikunin/ mice (which cannot assemble I
I) (Zhuo
et al., 2001a) and Tnfaip6/ mice
(Fulop et al., 2003
) are
infertile because of instability of the cumulus matrix and lack of oocyte
fertilization, like Ptx3/ mice. Co-operative
interaction between TNFAIP6 and I
I has been demonstrated. In the
expanding cumulus the heavy chains of I
I are covalently transferred
from the chondroitin sulfate to HA (Chen et
al., 1996
), and TNFAIP6 is clearly essential for completing this
coupling reaction, as heavy chain-HA complexes do not form in
Tnfaip6-deficient mice (Fulop et
al., 2003
). In addition, a covalent complex between one TNFAIP6
molecule and one of the heavy chains is also formed, and accumulates in the
cumulus matrix (Mukhopadhyay et al.,
2001
). It has been proposed that HA-linked heavy chains and
TNFAIP6-heavy chain complexes stabilize the cumulus matrix by cross-linking
separate HA molecules (Chen et al.,
1992
; Chen et al.,
1996
; Zhuo et al., 2001a;
Fulop et al., 2003
). Here, we
demonstrate that both types of complexes are present in the
Ptx3/ cumuli. This indicates that, although
necessary, these complexes are not sufficient to confer stability to the
cumulus matrix and that the PTX3 matrix-stabilizing activity is exerted
through an independent mechanism. In this regard, PTX3 binds TNFAIP6 (through
a site that is distinct from its HA-binding surface) and could therefore serve
as an additional way of cross-linking the matrix via the association of
TNFAIP6 with HA. PTX3, as TNFAIP6 (Carrette
et al., 2001
), co-localizes with HA throughout the matrix, from
the periphery of the cumulus to the zona pellucida. As PTX3 is unable to bind
to or form covalent bonds with HA, TNFAIP6 is the likely matrix component
involved in mediating such interaction. PTX3 is predominantly assembled as a
large multimer complex consisting of two decamers
(Bottazzi et al., 1997
), and
competition-binding studies suggest that each protomer can bind an individual
TNFAIP6 molecule. As illustrated in Fig.
10, PTX3/TNFAIP6 complexes might thus serve as an anchoring site
for multiple HA molecules, thereby substantially strengthening and stabilizing
the HA network. HA-protein interaction is crucial for the formation and
stability of extracellular matrix in several tissues in both physiological and
pathological conditions (Day and
Prestwich, 2002
; Tammi et al.,
2002
). The finding that the long pentraxin PTX3 is a component of
the extracellular matrix of the cumulus oophorus, essential for HA
organization, raises the likely possibility of a similar localization and
function of this molecule in certain HA-enriched inflammatory tissues, such as
occur in rheumatoid arthritis, where TNFAIP6 is also expressed
(Milner and Day, 2003
).
|
Finally, the primary structure of PTX3 is conserved between mouse and
humans (Introna et al., 1996),
and we report that PTX3 is also expressed in the human periovulatory cumulus
oophorus, and that it is present in the cumulus matrix as well. Therefore, it
is likely that PTX3 may play the same role in human, as it does in mouse
female fertility. This implies that PTX3 deficiency might be a cause of
unexplained infertility in women. Based on the
Ptx3/ mouse model, PTX3-deficient women
would be infertile despite a normal ovulation. Hormonal therapy and/or
artificial insemination would be ineffective in overcoming pregnancy failure
in these women, and in vitro fertilization should be considered the
first-choice treatment for them.
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ACKNOWLEDGMENTS |
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Footnotes |
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