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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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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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 (I
I) and
pre-
-trypsin inhibitor (P
I) 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 I
I (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 I
I (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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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 I
I (Dako). Mouse sera with or
without chondroitinase ABC digestion were used as a reference for the size of
I
I, P
I 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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DISCUSSION |
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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 P
I and I
I
(Salier et al., 1996
). These
complex serum proteins also contain one or two evolutionarily-related heavy
chains (HC1 and HC2 in I
I, and HC3 in P
I). 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 I
I and P
I
(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
).
|
We have shown recently that Tnfip6 forms covalent complexes with HC1 and
HC2 of II (but not with HC3 of P
I) 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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