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doi: 10.1242/10.1242/dev.00422
DEVELOPMENT AND DISEASE |
1 Section of Connective Tissue Biology, Department of Biomedical Engineering,
The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA
2 Section of Biochemistry and Molecular Biology, Departments of Orthopedic
Surgery and Biochemistry, Rush University at Rush-Presbyterian-St. Luke's
Medical Center, Chicago, Illinois 60612, USA
3 MRC, Immunochemistry Unit, Department of Biochemistry, University of Oxford,
OX1 3QU Oxford, UK
4 Department of Public Health and Cell Biology, University of Rome `Tor
Vergata', 00133 Rome, Italy
* Author for correspondence (e-mail: fulop{at}bme.ri.ccf.org)
Accepted 23 January 2003
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-trypsin inhibitor family. As a
consequence, TNFIP6-deficient females are sterile. Cultured TNFIP6-deficient
cumulus cell-oocyte complexes also fail to expand when stimulated with
dibutyryl cyclic AMP or epidermal growth factor. Recombinant TNFIP6 is able to
catalyze the covalent transfer of heavy chains to hyaluronan in a cell-free
system, restore the expansion of Tnfip6-null cumulus cell-oocyte
complexes in vitro, and rescue the fertility in Tnfip6-null females.
These results provide clear evidence that TNFIP6 is a key catalyst in the
formation of the cumulus extracellular matrix and indispensable for female
fertility.
Key words: Cumulus cell, Tumor necrosis factor-induced protein-6, Tnfip6, Tsg6, Inter--trypsin inhibitor, Hyaluronan, Extracellular matrix, Infertility
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INTRODUCTION |
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SUMMARY
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MATERIALS AND METHODS
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DISCUSSION
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).
One of the pivotal processes in ovulation is the cumulus mucification, i.e.
the synthesis and assembly of a highly viscoelastic extracellular matrix by
the cumulus cells (Salustri et al.,
1993). Several studies have shown that this extracellular matrix
is crucial for female fertility (Chen et
al., 1993; Hess et al.,
1999; Zhuo et al.,
2001). Although integral components of this matrix have been
identified, the mechanism of matrix assembly is not completely understood.
Hyaluronan, a negatively charged glycosaminoglycan, is the major component
of the COC matrix, and is responsible for the dramatic expansion of the COC
after the midcycle luteinizing hormone (LH) surge
(Salustri et al., 1992). The
heavy chains (HCs) of inter--trypsin inhibitor (II) and
pre--trypsin inhibitor (PI) are also specifically incorporated
into this matrix (Chen et al.,
1992), and knockout studies have identified a crucial role for
these molecules in COC matrix assembly and female fertility
(Zhuo et al., 2001;
Sato et al., 2001). A pivotal
step of the matrix assembly seems to be the covalent transfer of these HCs to
hyaluronan through a transesterification process
(Chen et al., 1996). It has
been suggested that these heavy chains could crosslink hyaluronan through
covalent and ionic bonds and stabilize the mucified COC matrix
(Chen et al., 1994;
Chen et al., 1996).
We and others have recently demonstrated that the gene for tumor necrosis
factor-induced protein-6 (Tnfip6; also known as tumor necrosis
factor-stimulated gene-6, Tsg6) is specifically expressed in COCs
undergoing mucification (Fulop et al.,
1997; Yoshioka et al.,
2000; Varani et al.,
2002), and that TNFIP6 is incorporated into the COC matrix
(Mukhopadhyay et al., 2001;
Carrette et al., 2001). The LH
surge also induces Tnfip6 expression in granulosa cells, and the
oocyte-derived growth differentiation factor-9 has been implicated in this
process (Yoshioka et al.,
2000; Varani et al.,
2002). Thus, the synthesis of TNFIP6 appears to be under both
autocrine and paracrine controls. The translated product of Tnfip6 is
an approx. 37 kDa glycoprotein (Lee et
al., 1992) that is able to specifically bind hyaluronan
(Kohda et al., 1996;
Parkar and Day, 1997) and can
form a complex with II (Wisniewski
et al., 1994; Mukhopadhyay et
al., 2001; Nentwich et al.,
2002). TNFIP6 is usually expressed under inflammatory conditions
(Wisniewski et al., 1993), and
its anti-inflammatory and chondroprotective effects in arthritis are well
documented (Wisniewski et al.,
1996; Bardos et al.,
2001; Glant et al.,
2002). However, in the expanding COCs, it has been suggested that
TNFIP6 plays the role as an extracellular matrix organizer by interacting with
both hyaluronan and II (Fulop et
al., 1997).
In this study we demonstrate that TNFIP6 is the catalyst that covalently
transfers the heavy chains of the II-related proteins onto hyaluronan.
TNFIP6-deficient mice are unable to complete this biochemical reaction, the
females fail to assemble their cumulus extracellular matrix and consequently
become sterile.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). A 10.7
kb genomic DNA fragment was isolated from a P1 clone (P-2199; Incyte Genomics)
by HindIII digestion (Fig.
1A). This fragment contained the entire promoter region, exon 1
and a 5.2 kb fragment of intron 1. The 5.5 kb long 5' arm of the genomic
fragment was inserted upstream of the PGK promoter-neor-poly(A)
cassette of expression vector pPNT
(Tybulewicz et al., 1991),
into the NdeI cleavage site of exon 1, disrupting the reading frame
and creating a stop codon 94 bp downstream of the translation start site. The
5.2 kb 3' arm genomic fragment was cloned downstream of the Neo
cassette. Linearized vector was electroporated into embryonic stem (ES) cells
of 129S6/SvEvTac origin (TC-1). Over 150 ES cell clones were identified after
positive and negative selection. Homologous integration was detected in three
ES cell clones, which were karyotyped and then clone no. 135
(Fig. 1B) was injected into
C57Bl/6 blastocysts. Two chimeric founder males were obtained and mated with
wild-type BALB/c females to establish two independent colonies. Since female
Tnfip6–/– mice were infertile, the colonies
were maintained by mating Tnfip6+/– or
Tnfip6–/– males with BALB/c females.
Tnfip6–/– females were generated by
intercrossing Tnfip6+/– females and
Tnfip6–/– males. All mice were genotyped for
the presence of the Neo gene and further tested for hetero- or
homozygozity using gene-specific primers
(Fig. 1C and
Table 1). Embryonic fibroblast
cells were isolated from wild-type, heterozygous and TNFIP6-deficient E18.5
embryos. Tnfip6-specific mRNA expression and protein secretion were
confirmed in non-stimulated and TNF-stimulated fibroblast cultures
(Fig. 1D,E).
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) which
is 94% identical to the murine protein] was expressed in Schneider 2 cells
[derived from Drosophila melanogaster
(Schneider, 1972)], purified
using ion exchange and reverse-phase high performance liquid chromatography,
and the protein concentration was determined by amino acid analysis as
described previously (Nentwich et al.,
2002). Mouse recTNFIP6 was purified on a Hyaluronan-Sepharose
column from supernatants of Chinese hamster ovary cells (CHO-K1; American Type
Culture Collection) stable-transfected with Lonza pEE14.1 vector (Lonza
Biologics plc) containing a full-length (1654 bp) mouse Tnfip6 cDNA
clone as described previously (Bardos et
al., 2001). Purified recTNFIP6 was tested by western blot using a
rabbit polyclonal antibody (TSG-6-CR21) raised against a synthetic peptide
(135CGGVFTDPKRIFKSPG) located at the N-terminal end of the CUB
domain and quantitated with sandwich enzyme-linked immunosorbent assay as
described previously (Bardos et al.,
2001).
Induction of ovulation
Superovulation was induced by consecutive injections of gonadotropins.
First, 21-day old female mice were injected intraperitoneally with 5 U
pregnant mares' serum gonadotropin (PMSG, Sigma) in 100 µl
phosphate-buffered saline (PBS) to induce follicle maturation. Ovulation was
induced by intraperitoneal injection of 5 U human chorionic gonadotropin (hCG,
Sigma) in 100 µl PBS 48 hours after the PMSG injection.
In vivo fertilization
Superovulation was induced as described above, and the females were paired
with wild-type males 2 hours after the hCG injection (at midnight). Females
were checked for vaginal plugs the next morning, and sacrificed 36 hours after
the hCG injection. Oocytes and two-cell embryos were collected from the
oviducts to assess in vivo fertilizability.
Isolation and culture of COCs
Ovaries and fallopian tubes were dissected in Minimum Essential Medium
(MEM) containing 25 mM Hepes, 0.1% bovine serum albumin and 50 ng/ml
gentamycin. Ovulated COCs were collected from the fallopian tubes 13-13.5
hours after the hCG injection. For cultures, ovaries were dissected from the
mice 48 hours after PMSG treatment, and COCs were isolated by puncturing the
larger follicles with a sterile needle. The isolated compact COCs were washed
once with standard COC culture medium (MEM supplemented with 5% fetal bovine
serum (FBS), 3 mM glutamine, 0.3 mM sodium pyruvate and 50 ng/ml gentamycin),
then cultured in standard COC culture medium in the absence or the presence of
either epidermal growth factor (EGF, 3 ng/ml; Sigma) or dibutyryl cyclic AMP
(dbcAMP, 1 mM; Sigma) at 37°C for 18 hours. In certain cases, COCs from
Tnfip6–/– females were cultured in the
presence of 1 µg/ml human recTNFIP6 protein to restore cumulus
mucification. Matrix expansion was monitored morphologically using an Olympus
inverted microscope.
Immunohistochemistry
Ovaries were harvested, fixed in 4% paraformaldehyde, and embedded in
paraffin. Deparaffinized sections were blocked in Hank's balanced salt
solution (HBSS) containing 2% FBS at room temperature for 1 hour and stained
with biotinylated hyaluronan binding protein (HABP, 5 µg/ml; Seikagaku) in
HBSS, 2% FBS at 4°C for 16 hours. Fluorescein isothiocyanate-conjugated
streptavidin (2 ng/ml; Vector Laboratories) was used as a secondary conjugate
in HBSS, 2% FBS at 25°C for 1 hour. The slides were washed in HBSS and
mounted with Vectashield containing DAPI (Vector Laboratories). Sections were
visualized with a confocal microscope (Leica TCS-SP). Hematoxylin and Eosin
staining was performed using standard procedures.
Protein extraction from ovaries
The ovulatory process was induced as described above, and mice were
sacrificed 10 hours after the hCG injection. Dissected ovaries were minced and
digested with 200 mU Streptomyces hyaluronidase in 100 µl PBS at 37°C
for 24 hours in the presence of protease inhibitors (5 mM EDTA, 1 mM
iodoacetamide, 5 µg/ml pepstatin A, 5 µg/ml aprotinin, 5 µg/ml
leupeptin and 10 mM benzamidine). Each digestion mixture, including the tissue
debris, was dried in a Speed-Vac concentrator and resuspended in reducing
Laemmli-buffer (Laemmli,
1970). Samples were analyzed by SDS-PAGE and western blotting
using a polyclonal antibody against II (Dako). Mouse sera with or
without chondroitinase ABC digestion were used as a reference for the size of
II, PI and their HCs.
TNFIP6-mediated transfer of the heavy chains of II family
members to hyaluronan
High molecular mass hyaluronan (12 µg, mol. mass: 5000 kDa; Healon GV,
Pharmacia & Upjohn) and mouse serum (10 µl, Rockland Immunochemicals)
were incubated in the absence or presence of human recTNFIP6 (500 ng) in 100
µl PBS at 37°C for 24 hours. A 10 µl aliquot of each reaction
mixture was further digested with 200 mU Streptomyces hyaluronidase at
37°C for 1 hour. The transfer of the heavy chains was monitored by Western
blot analysis as described above.
Rescue of female fertility in vivo
The rescue of fertility in Tnfip6–/–
females was attempted by two different approaches. First,
Tnfip6–/– female mice were superovulated as
described above and also received 100 µg of purified mouse recTNFIP6
protein intraperitoneally together with the hCG injection (total volume of 100
µl). Stud males were transferred into the female cages 6 hours later.
Vaginal plugs were identified next morning. A second rescue approach was
performed using in vivo genetic manipulations by generating
Tnfip6–/– females that also carried the
Tnfip6 transgene. Initially, transgenic females
(Tnfip6-Tg+/+), constitutively expressing Tnfip6
(Glant et al., 2002), were
mated with Tnfip6–/– males, first to generate
heterozygous
Tnfip6+/–Tnfip6-Tg+/–
offsprings and then to select females lacking the wild-type Tnfip6
gene but having the transgene. Although the Tnfip6-Tg expression
vector (pSP/44-3) was designed to drive cartilage-specific expression
(Glant et al., 2002), recently
we found that the construct is `leaking' and all tissues and organs, including
ovaries, constitutively express some levels of transgene-derived
Tnfip6 (data not shown).
Tnfip6–/–Tnfip6-Tg+/–
or Tnfip6–/–Tnfip6-Tg+/+
females without superovulation were mated with wild-type stud males.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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stimulation
(Fig. 1E). These results
clearly showed the absence of TNFIP6 mRNA and protein in
Tnfip6–/– fibroblasts
(Fig. 1D,E).
Tnfip6–/– mice were normal at birth and showed
normal growth. The only apparent phenotypic abnormality was the sterility of
the Tnfip6–/– females
(Table 2).
Tnfip6+/– females and
Tnfip6–/– males were fertile, and the average
litter sizes in all combinations were highly comparable
(Table 2). In order to
determine if the sterility of Tnfip6–/–
females resulted from their inability to ovulate, females of the three
Tnfip6 genotypes were induced to superovulate by consecutive
injections of PMSG and hCG. Oocytes were recovered from the fallopian tubes
13.5 hours after hCG injection and counted. Although
Tnfip6–/– females were able to ovulate, the
average number of oocytes recovered from their oviducts after superovulation
(14±2, n=8) was significantly lower (P<0.001) than
the average recovered from their wild-type (29±10, n=8) or
heterozygous littermates (32±12, n=12). In addition, sexually
mature (12-week old) Tnfip6–/– females also
appeared to be able to ovulate under normal endocrine conditions as evidenced
by the presence of corpus lutea in their ovaries
(Fig. 2A-C). In order to
investigate if oocytes from Tnfip6–/– females
can be fertilized in vivo, superovulated females were mated with wild-type
males, and the ability of the oocytes to reach the two-cell stage was
assessed. While the vast majority (>90%) of oocytes from wild-type and
Tnfip6+/– females successfully reached the two-cell
stage in these assays, we were unable to detect any two-cell embryos in
Tnfip6–/– females
(Fig. 3). Thus, the sterility
of the Tnfip6–/– females was the result of the
inability of their oocytes to be fertilized in vivo.
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;
Hess et al., 1999;
Zhuo et al., 2001), and that
TNFIP6 specifically incorporates into this matrix during the ovulatory process
(Mukhopadhyay et al., 2001;
Carrette et al., 2001). Thus,
one possible explanation for the sterility of the
Tnfip6–/– females is that the COCs do not
mucify correctly, which in turn could lead to the infertilizability of the
oocyte. To explore this possibility, female mice were superovulated, and
cumulus matrix expansion was evaluated using histochemistry. Preovulatory
follicles formed 48 hours after the PMSG injection, contained compact COCs in
all three Tnfip6 genotypes and were indistinguishable
(Fig. 4A-C). However, when
cumulus mucification was induced by hCG injection, the morphology of the
homozygous preovulatory follicles differed dramatically from their wild-type
and heterozygous counterparts. While wild-type and
Tnfip6+/– females demonstrated normal cumulus
expansion 10 hours after hCG injection,
Tnfip6–/– females failed to do so
(Fig. 4D-F). Instead, oocytes
in the homozygotes shed a large number of cumulus cells at this stage. The few
cumulus cells retained around the oocyte did not show any signs of expansion
(Fig. 4F), suggesting defects
in cumulus matrix organization and stability. These observations were further
supported when ovarian sections were stained for hyaluronan, the major
structural component of the mucified cumulus extracellular matrix. Wild type
and heterozygous females showed intense hyaluronan staining in their cumulus
matrix 10 hours after hCG injection (Fig.
4G,H). In contrast, homozygous littermates had only faint
hyaluronan staining at this stage, indicating the lack of the normal cumulus
matrix in this genotype (Fig.
4I). High power magnification of sections revealed that the
detectable hyaluronan staining in Tnfip6–/–
mice was associated with the cell surface of the cumulus cells and with the
follicular fluid (Fig. 4L).
These observations suggested that the cumulus cells of the
Tnfip6–/– mice still synthesized hyaluronan
but were unable to organize it into an extracellular matrix.
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; Eppig,
1981; Downs, 1989).
In order to study the ability of TNFIP6-deficient COCs to mucify in vitro,
COCs were isolated from PMSG-primed female mice and cultured for 18 hours in
the absence or presence of either EGF or dbcAMP. In the untreated cultures,
the COCs from all three Tnfip6 genotypes remained compact and
morphologically indistinguishable (Fig.
6A-C). While both EGF and dbcAMP were able to induce expansion of
COCs from wild-type and Tnfip6+/– females
(Fig. 6D,E,G,H), COCs from
Tnfip6–/– females shed most of their cumulus
cells (Fig. 6F,I), which
closely resembled the phenotype observed in vivo
(Fig. 4F). Culturing
TNFIP6-deficient COCs in the presence of recTNFIP6 successfully restored their
ability to expand (Fig. 6J,K), further supporting the crucial role of this protein in cumulus matrix
organization.
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I family members onto hyaluronanI-related
molecules diffuse from the blood into the preovulatory follicle
(Powers et al., 1995), and
that the covalent transfer of the HCs of these molecules onto hyaluronan was
crucial for the formation of the COC extracellular matrix
(Zhuo et al., 2001). In order
to investigate the presence of hyaluronan-HC covalent complexes in the ovaries
of the three Tnfip6 genotypes, we digested the ovaries (10 hours
after hCG injection) with Streptomyces hyaluronidase, an enzyme highly
specific for hyaluronan. Western blot analysis revealed that ovaries from all
three genotypes contained II and PI. However, hyaluronan-linked
HCs could only be detected in wild-type and heterozygous mice
(Fig. 7A). Hyaluronidase
extracts of homozygous ovaries lacked the 
85 kDa band (representing
single heavy chains) and additional high molecular mass bands
(Fig. 7A, open arrowheads).
These latter bands most likely represent dimers and clusters of HCs located
close to each other on a single hyaluronan chain. The close clustering of
these chains would render resistance of these structures to hyaluronidase
digestion as suggested by Chen et al. (Chen
et al., 1996). In order to investigate whether TNFIP6 was
necessary for the transfer of the HCs onto hyaluronan, we incubated hyaluronan
with mouse serum (the physiological source of II and PI) in the
absence or presence of recTNFIP6. Covalent transfer of the HCs to hyaluronan
did not occur in the absence of recTNFIP6
(Fig. 7B, lanes 1 and 2).
However, when recTNFIP6 was added to the reaction mixture, the II and
PI bands disappeared, and a new band immunoreactive with anti-II
antibody was detected at the top of the gel indicating the formation of the
high molecular mass hyaluronan-HC complexes
(Fig. 7B, lane 3). These
complexes could not be dissociated in reducing Laemmli buffer, but could be
readily broken down by Streptomyces hyaluronidase
(Fig. 7B, lane 4), implying the
covalent nature of the hyaluronan-HC complex.
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) in
otherwise Tnfip6–/– females, we found
significantly higher litter sizes (Table
2). These results indicate that the introduction of TNFIP6, either
as a recombinant protein or as the product of the Tnfip6 transgene,
can rescue fertility in Tnfip6–/– females. |
DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Yoshioka et al., 2000;
Mukhopadhyay et al., 2001;
Carrette et al., 2001). Thus,
TNFIP6 does not seem to have a crucial role in follicular development up to
the preovulatory stage. However, TNFIP6 has a pivotal importance in the
mucification of the COC during the preovulatory period. The COC extracellular
matrix fails to assemble in TNFIP6-deficient mice in vivo, as visualized by
the lack of hyaluronan incorporation and ensuing COC expansion. Most of the
cumulus cells detach from the COCs just prior to the follicular rupture and
disperse into the follicular space. This observation suggests that the
extracellular matrix is absolutely necessary to maintain the cumulus mass in
the oocyte's vicinity once the contacts between cumulus cells are severed.
Similar conclusions can be drawn from the lack of cumulus matrix expansion
after stimuli by either EGF or dbcAMP (Fig.
6), potent initiators of COC expansion in vitro
(Downs, 1989;
Salustri et al., 1990).
Tnfip6–/– female mice have markedly lower
number of oocytes in their oviducts after superovulation than their wild-type
or heterozygous littermates. This difference could be the result of (i) lower
number of preovulatory follicles, (ii) lower rate of ovulation or (iii)
inefficient pickup of the ovulated oocytes by the fimbria. Although we did not
compare the actual number of preovulatory follicles, the morphology of these
follicles was very similar among the three genotypes. In addition,
Tnfip6 is expressed only after the LH surge (i.e., after preovulatory
follicle formation) (Fulop et al.,
1997) and is not expected to influence the number of preovulatory
follicles. Lower numbers of COCs (or nude oocytes) in the oviducts have also
been demonstrated in other experimental animal models in which COC expansion
was inhibited, such as during 6-diazo-5-oxo-1-norleucine treatment
(Chen et al., 1993), hyaluronan
oligosaccharide treatment (Hess et al.,
1999), and in the bikunin knockout mice
(Zhuo et al., 2001;
Sato et al., 2001). While
these studies have resulted in the speculation that the reduced number of
oocytes in the oviducts is due to the lower ovulation rate, inefficient
fimbrial pickup cannot be excluded
(Mahi-Brown and Yanagimachi,
1983). In fact, polycationic macromolecules, such as poly-L-lysine
have been shown to inhibit COC transport along the fallopian tube
(Norwood et al., 1978),
suggesting that the negatively charged hyaluronan-rich COC matrix may play an
important role in this process. The lack of the hyaluronan-rich COC matrix
(such as in Tnfip6–/– mice) could result in
less efficient pickup of the nude oocytes by the ciliary structures of the
fimbria.
The observed phenotype of Tnfip6–/– females
is similar to that previously described in bikunin-null females
(Zhuo et al., 2001). Bikunin
is the light chain component of PI and II
(Salier et al., 1996). These
complex serum proteins also contain one or two evolutionarily-related heavy
chains (HC1 and HC2 in II, and HC3 in PI). The polypeptide
chains are covalently linked together by a single chondroitin sulfate chain
through ester and glycosidic bonds (for schematic representation see
Fig. 8). Bikunin null mice are
unable to assemble II and PI
(Zhuo et al., 2001). Although
unprocessed HCs are present in the sera of these animals, these HCs cannot be
covalently transferred to hyaluronan in the expanding COCs. The absence of
this biochemical reaction leads to impaired cumulus mucification, ovulation of
nude oocytes, and female sterility (Zhuo
et al., 2001).
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I-null and PI-null) female mice suggest
a cooperative role of TNFIP6 and the HCs of the II family members in
the formation of the COC extracellular matrix
(Fig. 8). After the midcycle LH
surge, cumulus cells start to synthesize hyaluronan, while both cumulus and
granulosa cells synthesize TNFIP6
(Yoshioka et al., 2000;
Mukhopadhyay et al., 2001;
Carrette et al., 2001;
Varani et al., 2002). These
processes are accompanied by the diffusion of the II family members
into the preovulatory follicle (Powers et
al., 1995). Our present data provide the first evidence that
TNFIP6 facilitates the covalent transfer of HCs from II and PI
to hyaluronan. These HCs can stabilize the COC extracellular matrix by
crosslinking separate hyaluronan chains through covalent and ionic
interactions as hypothesized before (Fig.
8) (Chen et al.,
1994; Chen et al.,
1996). According to our model, deficiencies in either TNFIP6 or
II (and PI) result in the same phenotype, i.e. lack of cumulus
matrix formation. A previous study by Chen et al.
(Chen et al., 1996) suggested
that the factor that facilitates HC transfer to hyaluronan is produced by
granulosa cells. While granulosa cells are potent sites of TNFIP6 synthesis
(Yoshioka et al., 2000;
Mukhopadhyay et al., 2001;
Carrette et al., 2001;
Varani et al., 2002), TNFIP6
synthesized by cumulus cells alone is clearly sufficient to form the
extracellular matrix as demonstrated by COC cultures in the absence of
granulosa cells (Fig. 6).
However, granulosa cell-derived TNFIP6 can substantially strengthen the
stability of the COC matrix, since the preovulatory follicle contains 50
times more granulosa cells than cumulus cells
(Pedersen, 1970;
Salustri et al., 1992).
Indeed, COCs expanded in vivo have been shown to be 6 times more
resistant to mechanical forces than those expanded in vitro
(Chen et al., 1996).
We have shown recently that Tnfip6 forms covalent complexes with HC1 and
HC2 of II (but not with HC3 of PI) in the COC matrix, and that
these complexes, similarly to the single HCs, are very tightly associated with
hyaluronan in the COC matrix (Mukhopadhyay
et al., 2001). We have hypothesized that these HC-TNFIP6 complexes
play a role in the stabilization of the COC matrix by crosslinking separate
hyaluronan chains. Our current findings now further suggest that HC-TNFIP6
complexes could be intermediates or by-products during the TNFIP6-facilitated
HC transfer to hyaluronan (Fig.
8). This hypothesis is favored by our previous observation that
only a small portion of the HCs are present in the form of HC-TNFIP6 complexes
in the COC matrix (Mukhopadhyay et al.,
2001). The small amount of HC-TNFIP6 complexes, however, could
still significantly contribute to matrix stabilization as a result of the very
strong hyaluronan-binding affinity of TNFIP6
(Kohda et al., 1996;
Parkar and Day, 1997).
Therefore, the cumulus matrix is more likely stabilized by multiple crosslinks
involving individual HCs and HC-TNFIP6 complexes
(Fig. 8).
Our studies present clear evidence that correct mucification of the cumulus
mass is an obligatory step for female fertility. Successful in vitro
fertilization has been previously correlated with the degree of cumulus matrix
formation (Chen et al., 1993),
and complete failure of cumulus expansion results in the lack of
fertilizability of the oocytes in bikunin-null
(Zhuo et al., 2001) and
pentraxin-3-null (Varani et al.,
2002) female mice. Similar to these animal models, TNFIP6
deficiency also causes impaired cumulus matrix formation, the ovulation of
nude oocytes and the lack of fertilizability of these oocytes.
Our findings may lead to new approaches for the treatment of certain women with unexplained infertility. TNFIP6 deficiency could be a cause in a group of these women, and genetic screening should be applied to identify this possibility. TNFIP6-deficient women are not expected to respond to gonadotropin therapy (as shown in our animal model); rather a treatment with recombinant TNFIP6 should be developed. Our in vitro and in vivo rescue experiments lend feasibility to this approach.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Bardos, T., Kamath, R. V., Mikecz, K. and Glant, T. T.
(2001). Anti-inflammatory and chondroprotective effect of TSG-6
(tumor necrosis factor-alpha-stimulated gene-6) in murine models of
experimental arthritis. Am. J. Pathol.
159,1711
-1721.
Carrette, O., Nemade, R. V., Day, A. J., Brickner, A. and
Larsen, W. J. (2001). TSG-6 is concentrated in the
extracellular matrix of mouse cumulus oocyte complexes through hyaluronan and
inter-alpha-inhibitor binding. Biol. Reprod.
65,301
-308.
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M. S. Rugg, A. C. Willis, D. Mukhopadhyay, V. C. Hascall, E. Fries, C. Fulop, C. M. Milner, and A. J. Day Characterization of Complexes Formed between TSG-6 and Inter-{alpha}-inhibitor That Act as Intermediates in the Covalent Transfer of Heavy Chains onto Hyaluronan J. Biol. Chem., July 8, 2005; 280(27): 25674 - 25686. [Abstract] [Full Text] [PDF]
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R. A. Dragovic, L. J. Ritter, S. J. Schulz, F. Amato, D. T. Armstrong, and R. B. Gilchrist Role of Oocyte-Secreted Growth Differentiation Factor 9 in the Regulation of Mouse Cumulus Expansion Endocrinology, June 1, 2005; 146(6): 2798 - 2806. [Abstract] [Full Text] [PDF]
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C. D. Blundell, A. Almond, D. J. Mahoney, P. L. DeAngelis, I. D. Campbell, and A. J. Day Towards a Structure for a TSG-6{middle dot}Hyaluronan Complex by Modeling and NMR Spectroscopy: INSIGHTS INTO OTHER MEMBERS OF THE LINK MODULE SUPERFAMILY J. Biol. Chem., May 6, 2005; 280(18): 18189 - 18201. [Abstract] [Full Text] [PDF]
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J. S. Richards, I. Hernandez-Gonzalez, I. Gonzalez-Robayna, E. Teuling, Y. Lo, D. Boerboom, A. E. Falender, K. H. Doyle, R. G. LeBaron, V. Thompson, et al. Regulated Expression of ADAMTS Family Members in Follicles and Cumulus Oocyte Complexes: Evidence for Specific and Redundant Patterns During Ovulation Biol Reprod, May 1, 2005; 72(5): 1241 - 1255. [Abstract] [Full Text] [PDF]
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H.-G. Wisniewski, E. S. Snitkin, C. Mindrescu, M. H. Sweet, and J. Vilcek TSG-6 Protein Binding to Glycosaminoglycans: FORMATION OF STABLE COMPLEXES WITH HYALURONAN AND BINDING TO CHONDROITIN SULFATES J. Biol. Chem., April 15, 2005; 280(15): 14476 - 14484. [Abstract] [Full Text] [PDF]
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K. W. Sanggaard, H. Karring, Z. Valnickova, I. B. Thogersen, and J. J. Enghild The TSG-6 and I{alpha}I Interaction Promotes a Transesterification Cleaving the Protein-Glycosaminoglycan-Protein (PGP) Cross-link J. Biol. Chem., March 25, 2005; 280(12): 11936 - 11942. [Abstract] [Full Text] [PDF]
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H. Ashkenazi, X. Cao, S. Motola, M. Popliker, M. Conti, and A. Tsafriri Epidermal Growth Factor Family Members: Endogenous Mediators of the Ovulatory Response Endocrinology, January 1, 2005; 146(1): 77 - 84. [Abstract] [Full Text] [PDF]
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F. L Lopes, J. A Desmarais, and B. D Murphy Embryonic diapause and its regulation Reproduction, December 1, 2004; 128(6): 669 - 678. [Abstract] [Full Text] [PDF]
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L.J. McKenzie, S.A. Pangas, S.A. Carson, E. Kovanci, P. Cisneros, J.E. Buster, P. Amato, and M.M. Matzuk Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF Hum. Reprod., December 1, 2004; 19(12): 2869 - 2874. [Abstract] [Full Text] [PDF]
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E. Nagyova, A. Camaioni, R. Prochazka, and A. Salustri Covalent Transfer of Heavy Chains of Inter-{alpha}-Trypsin Inhibitor Family Proteins to Hyaluronan in In Vivo and In Vitro Expanded Porcine Oocyte-Cumulus Complexes Biol Reprod, December 1, 2004; 71(6): 1838 - 1843. [Abstract] [Full Text] [PDF]
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M. Shimada, M. Nishibori, Y. Yamashita, J. Ito, T. Mori, and J. S. Richards Down-Regulated Expression of A Disintegrin and Metalloproteinase with Thrombospondin-Like Repeats-1 by Progesterone Receptor Antagonist Is Associated with Impaired Expansion of Porcine Cumulus-Oocyte Complexes Endocrinology, October 1, 2004; 145(10): 4603 - 4614. [Abstract] [Full Text] [PDF]
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L. Zhuo, V. C. Hascall, and K. Kimata Inter-{alpha}-trypsin Inhibitor, a Covalent Protein-Glycosaminoglycan-Protein Complex J. Biol. Chem., September 10, 2004; 279(37): 38079 - 38082. [Full Text] [PDF]
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S. A. Pangas, C. J. Jorgez, and M. M. Matzuk Growth Differentiation Factor 9 Regulates Expression of the Bone Morphogenetic Protein Antagonist Gremlin J. Biol. Chem., July 30, 2004; 279(31): 32281 - 32286. [Abstract] [Full Text] [PDF]
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A. Salustri, C. Garlanda, E. Hirsch, M. De Acetis, A. Maccagno, B. Bottazzi, A. Doni, A. Bastone, G. Mantovani, P. B. Peccoz, et al. PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization Development, April 1, 2004; 131(7): 1577 - 1586. [Abstract] [Full Text] [PDF]
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L. Mittaz, D.L. Russell, T. Wilson, M. Brasted, J. Tkalcevic, L.A. Salamonsen, P.J. Hertzog, and M.A. Pritchard Adamts-1 Is Essential for the Development and Function of the Urogenital System Biol Reprod, April 1, 2004; 70(4): 1096 - 1105. [Abstract] [Full Text] [PDF]
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D. Mukhopadhyay, A. Asari, M. S. Rugg, A. J. Day, and C. Fulop Specificity of the Tumor Necrosis Factor-induced Protein 6-mediated Heavy Chain Transfer from Inter-{alpha}-trypsin Inhibitor to Hyaluronan: IMPLICATIONS FOR THE ASSEMBLY OF THE CUMULUS EXTRACELLULAR MATRIX J. Biol. Chem., March 19, 2004; 279(12): 11119 - 11128. [Abstract] [Full Text] [PDF]
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C. D. Blundell, D. J. Mahoney, A. Almond, P. L. DeAngelis, J. D. Kahmann, P. Teriete, A. R. Pickford, I. D. Campbell, and A. J. Day The Link Module from Ovulation- and Inflammation-associated Protein TSG-6 Changes Conformation on Hyaluronan Binding J. Biol. Chem., December 5, 2003; 278(49): 49261 - 49270. [Abstract] [Full Text] [PDF]
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S. A. Ochsner, A. J. Day, M. S. Rugg, R. M. Breyer, R. H. Gomer, and J. S. Richards Disrupted Function of Tumor Necrosis Factor-{alpha}-Stimulated Gene 6 Blocks Cumulus Cell-Oocyte Complex Expansion Endocrinology, October 1, 2003; 144(10): 4376 - 4384. [Abstract] [Full Text] [PDF]
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W. Yingsung, L. Zhuo, M. Morgelin, M. Yoneda, D. Kida, H. Watanabe, N. Ishiguro, H. Iwata, and K. Kimata Molecular Heterogeneity of the SHAP-Hyaluronan Complex: ISOLATION AND CHARACTERIZATION OF THE COMPLEX IN SYNOVIAL FLUID FROM PATIENTS WITH RHEUMATOID ARTHRITIS J. Biol. Chem., August 29, 2003; 278(35): 32710 - 32718. [Abstract] [Full Text] [PDF]
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C. M. Milner and A. J. Day TSG-6: a multifunctional protein associated with inflammation J. Cell Sci., May 15, 2003; 116(10): 1863 - 1873. [Abstract] [Full Text] [PDF]
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