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Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka 431-3192, Japan
Address all correspondence and requests for reprints to: Dr. Hiroshi Kobayashi, Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka 431-3192, Japan.
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Abstract |
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-trypsin
inhibitor family in the gonadotropin-stimulated cumulus cells. Link
protein, a glycoprotein found in cartilage, interacts specifically with
hyaluronic acid and stabilizes the binding of proteoglycan monomers to
hyaluronic acid to form aggregates. The aim of this study was to
investigate the expression of immunoreactive link protein during
follicle development in rats and in cumulus cells in culture by
immunohistochemistry and Western blot as well as by specific
enzyme-linked immunosorbent assay. Immunohistochemical analysis
revealed that the extracellular matrix of cumulus cells that were
morphologically at a stage of COC expansion were markedly stained for
link protein, whereas granulosa cells from immature follicles were not
stained. Cumulus cells deposited link protein into the extracellular
matrix in an in vitro culture system. The staining
intensity was negated by the treatment with hyaluronidase, suggesting
that the link protein is bound to hyaluronic acid. We have identified a
42-kDa immunoreactive link protein in rat ovary during the preovulatory
period and in COC extracts. Addition of FSH to the medium of cumulus
cells in culture supplemented with 10% FBS and oocyte-conditioned
medium resulted in an increased rate of link protein synthesis. This
work suggests that the cumulus cells synthesize the link protein that
may stabilize the binding of inter--trypsin inhibitor or dermatan
sulfate proteoglycan to hyaluronic acid to make up hyaluronic acid-rich
matrix aggregate. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A natural ligand for hyaluronic acid has been discovered and identified
as a 240-kDa inter--trypsin inhibitor (6, 7, 8, 9, 10, 11) and dermatan sulfate
proteoglycan (12). Inter--trypsin inhibitor is comprised of three
genetically different polypeptides: two heavy chains and one light
chain (urinary trypsin inhibitor), which is one of the Kunitz-type
protease inhibitors present in human serum and urine (13). Urinary
trypsin inhibitor inhibits various serine proteases, such as trypsin,
-chymotrypsin, granulocyte elastase, plasmin, cathepsin G, and
hyaluronidase (13). Several articles have been devoted to study of the
molecular mechanisms responsible for the initiation, progression, and
maintenance of hyaluronic acid-rich matrix on the cumulus cells,
demonstrating the coincident appearance of hyaluronic acid and
proteoglycans (inter--trypsin inhibitor and dermatan sulfate
proteoglycan) during follicle development (5, 6, 9, 12, 14). In the
last few years, several studies have been made of the molecular system
that controls the initiation and progression of COC expansion. These
findings are very similar to the biochemical evidence indicating that
the aggregates involving hyaluronic acid and proteoglycans are
important components of the extracellular matrix of cartilage (15).
Camaioni et al. (12) reported that the dermatan sulfate
proteoglycan and the approximately 46-kDa protein synthesized by the
cumulus cells form similar ternary complexes that are necessary for
retaining hyaluronic acid in the COC matrix and hence are required for
successful COC expansion. The approximately 46-kDa protein has the same
molecular size as the link protein that interacts with hyaluronic acid
and hyaluronic acid-binding proteoglycans to form stable ternary
complexes in a variety of extracellular matrexes.
Link protein is a glycoprotein found in all hyaline cartilage examined
to date (15). It interacts specifically with a decasaccharide segment
of hyaluronic acid (12, 16, 17, 18, 19). It also stabilizes the binding of
proteoglycan monomers to hyaluronic acid to form aggregates and appears
to interact with both proteoglycan and hyaluronic acid (20, 21, 22). It has
been established that proteins of the inter--trypsin inhibitor
family associate with hyaluronic acid directly, and so does hyaluronic
acid-binding protein, which is comprised of cartilage proteoglycan core
protein and link protein (5). Link protein binds simultaneously to the
hyaluronic acid-binding region of the proteoglycan molecule and
hyaluronic acid (23). We (5) and others (12) thus speculate that link
protein may stabilize the binding between hyaluronic acid and
inter--trypsin inhibitor or other extracellular matrix proteins,
such as dermatan sulfate proteoglycan, and influence the spacing of the
monomers along the hyaluronic acid filament during follicle
development. However, there is no information on the expression and
localization of the link protein molecule during follicle development
in rats. For a better understanding of the physiology of COC expansion,
analysis of the expression of link protein in individual cells would be
required.
In the present study we investigated the presence and synthesis of link protein in rat COC. To this end we prepared specific antibodies raised against bovine nasal cartilage link protein and investigated the localization of immunoreactive-link protein in PMSG-treated hCG-stimulated mature rat ovaries immunohistochemically. In addition, we report herein the identification and synthesis of a protein with the antigenic and electrophoretic characteristics of link protein in cumulus cell cultures.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparations of polyclonal antibodies raised against link
protein
Polyclonal antibodies against bovine nasal cartilage link
protein were prepared by intradermal injection of rabbits with 2 mg
purified link protein emulsified in Freund’s complete adjuvant. Four
weeks after the first injection, the rabbit was boosted with 1 mg
protein in incomplete Freund’s adjuvant and then was boosted at 4-week
intervals. The antiserum was specific for bovine nasal cartilage link
protein, but not for aggrecan, and had a 50% maximal binding at a
dilution of 1:10,000 in a specific ELISA. This antiserum was reactive
with both 42-kDa (ovary) and 45-kDa (cartilage) proteins in Western
blot assay. Affinity-purified IgG was prepared by mixing 3 ml antiserum
with 1 ml link protein-coupled Sepharose 4B overnight at 4 C. After
washing, the IgG was eluted with 100 mmol/liter glycine-HCl, pH 2.5.
The pH of the eluted fractions was immediately raised, and the IgG was
stored at -20 C.
Animals and tissue sections
Twenty-five-day-old immature female Wistar rats and 10-week-old
adult cyclic female Wistar rats were purchased from SLC (Shizuoka,
Japan). Rats were housed in a temperature-controlled room with a 12-h
light, 12-h dark schedule and were fed chow and water ad
libitum. Female rat estrous cycles were monitored by daily vaginal
cytology. Only animals exhibiting two or more consecutive 4-day estrous
cycles were used in these experiments. We used animals taken during the
morning of estrous. The estrous cycle of adult rats was confirmed by
vaginal smear at 0900 h, and ovaries were obtained at 1500 h.
Immature rats were treated with ip injection of 15 IU PMSG in 0.2 ml
PBS, pH 7.4. Rats were treated with ip injection of 15 IU hCG 48 h
later, and ovaries were removed 12 h later and used for further
immunological (by Western blot) and immunohistochemical
experiments.
Isolation and culture of COC
For culture of cumulus cells, rats were killed 48 h after
PMSG injection, and the ovaries were transferred to HEPES-buffered HBSS
(Sigma Chemical Co., St. Louis, MO) containing 1%
(wt/vol) BSA. Large follicles were immediately punctured with a
27-gauge needle, and escaping COCs were harvested. Culture medium was
prepared according to the description of Singh et al. (26)
with few modifications and consisted of medium 199 with Earle’s salt
(Life Technologies, Grand Island, NY) supplemented with 25
mM HEPES buffer (Life Technologies), 2.5
mM glucosamine, 3 mM glutamine (Life Technologies), 0.03 mM sodium pyruvate (Life Technologies), 2.5 mM sodium lactate, 5
mM glucose (Yoneyama Chemical Co., Tokyo, Japan), 100 U/ml
penicillin, 100 µg/ml streptomycin, 0.25 mg/ml
Amphotericin (Life Technologies; further
referred to as medium), and 10% FBS (Life Technologies)
in the presence or absence of 10 µg/ml of FSH-LH (Sankyo Co. Ltd., Tokyo, Japan). Intact COCs were incubated in medium
containing 10% FBS and 10 µg/ml FSH for 12 h at 37 C.
In a parallel experiment, oocytes were stripped of granulosa cells by pipetting with a glass capillary. Granulosa cells were recovered by centrifugation at 20 x g for 5 min. The supernatant was discarded, and the cells were resuspended in culture medium. Furthermore, oocyte-conditioned medium was obtained by culturing isolated oocytes as described previously (27). Granulosa cells were suspended in the medium containing oocyte-conditioned medium (10 µl) at a final concentration of about 5 x 104 cells/100 µl supplemented with and without 10 µg/ml FSH. Fifteen oocytes were used to condition the 10 µl medium. Incubations were carried out for 12, 24, and 36 h at 37 C in an atmosphere of 95% air and 5% CO2.
Immunodetection of link protein in extracts from whole ovaries and
COC lysate
For immunodetection, whole ovaries of both adult cyclic rats and
mature PMSG-treated hCG-stimulated rats as well as cultured COCs were
homogenized in lysis buffer [8 M urea and 50
mM sodium acetate at pH 5.8 containing 50 U/ml
Streptomyces hyaluronidase (Calbiochem, La
Jolla, CA), 0.1% Triton X-100, 1 mmol/liter
phenylmethylsulfonylfluoride, 1 µg/ml leupeptin, and 1 µg/ml
pepstatin]. The protein content of the preparation was determined by
the method of Bradford (28), using BSA as the standard. Tissue extract
and COC lysate (50 µg) were mixed with the SDS sample buffer (5% SDS
and 10% glycerol), boiled for 5 min, separated by SDS-PAGE with 12%
gel according to Laemmli’s method (29), and electrophoretically
transferred onto polyvinylidine difluoride membranes (Immobilon,
Millipore Corp., Bedford, MA). The membranes were blocked
for 1 h in Tris-buffered saline (TBS) with 2% BSA and incubated
for 2 h with a 1:500 dilution of polyclonal antibodies raised
against link protein and then for 1 h with biotinylated goat
antirabbit IgG as the second antibodies (2 µg/ml, 1 h, 23 C;
DAKO Corp. Copenhagen, Denmark), followed by
avidin-peroxidase (2 µg/ml, 1 h, 23 C; DAKO Corp.).
Bands were visualized with the enhanced chemiluminescence detection
system (Amersham, Tokyo, Japan). In all experiments, some
strips were incubated with nonimmune rabbit IgG as a negative
control.
Immunohistochemistry
Culture of cumulus cells for immunohistochemistry: Granulosa
cells were seeded into Lab-Tek culture slides (Nunc, Copenhagen,
Denmark) at a density of approximately 1 x 104
cells/chamber in the medium containing oocyte-conditioned medium and
10% FBS supplemented with and without 10 µg/ml FSH-LH in triplicate
and cultured in a humidified atmosphere of 5% CO2 in air.
After 12-h incubation, the cells were fixed with 4% paraformaldehyde
in PBS and stained with polyclonal antibodies raised against link
protein, as performed in tissue sections (see below).
Rat ovaries were fixed in 4% (wt/vol) paraformaldehyde in 0.1 M PBS. The tissues were fixed at 23 C overnight and washed in 0.1 M phosphate buffer containing 0.25 M sucrose and 0.2 M glycine, pH 7.4, several times over a period of 16 h. After passing through a series of graded alcohol and xylene solutions, the tissues were embedded in paraffin by the standard procedures. Five-micron sections were taken on glass slides for immunostainings. In some cases the sections and the cells were treated with 50 U/ml Streptomyces hyaluronidase in 50 mM sodium acetate containing 0.15 M NaCl, pH 5.0, for 16 h at 23 C. Hematoxylin-stained sections, and cells were used for identification of follicles and cumulus cells.
Localization of link protein was examined immunohistochemically using the polyclonal antibodies for link protein. Deparaffinized and rehydrated tissue sections were immersed in 0.3% H2O2 in methanol to block endogenous peroxidase and were preincubated with 5% BSA in PBS for 1 h at 23 C to block nonspecific binding. The sections or cells were reacted with the antilink protein antibody diluted 1:200 (final concentration, 5 µg/ml) with 2% BSA in PBS for 16 h at 4 C in a humidified atmosphere. After washing in PBS three times for 15 min each time, the specimens were incubated with biotin-conjugated secondary antibodies (10 µg/ml; DAKO Corp.) diluted with 2% BSA in PBS for 1 h at 23 C. The specimens were then washed three times with PBS and incubated with avidin-peroxidase (DAKO Corp.) diluted 1:100 (10 µg/ml) with 2% BSA in PBS for 30 min at 23 C. They were washed three times with PBS. Peroxidase activity was seen after incubation in 100 mmol/liter TBS, pH 7.4, containing 0.03% H2O2 and 0.05% diaminobenzidine tetrahydrochloride. All sections were washed repeatedly with PBS and counterstained with hematoxylin. To ascertain whether link protein exists in association with hyaluronic acid, the sections of rat ovary were treated with 50 U/ml Streptomyces hyaluronidase in 50 mM sodium acetate containing 0.15 M NaCl, pH 5.0, for 16 h at 23 C. As a control, some of the sections were reacted with rabbit nonimmune IgG in place of the specific antibodies.
ELISAs for link protein
For the determination of link protein antigen, cell
culture conditions were continued for 12, 24, and 36 h in medium
containing oocyte-conditioned medium supplemented with and without 10
µg/ml FSH-LH in triplicate, and one flask was kept for cell counting.
After 12-, 24-, or 36-h incubation, the media were collected for ELISA
(see below) and stored at -20 C.
Immobilization of polyclonal antibody against link protein (2 µg/ml) to microtiter plates was carried out in 50 mM sodium carbonate buffer, pH 9.5, overnight at 4 C. All subsequent additions in ELISA were performed for 2 h at 23 C in TBS containing 2% BSA. After each addition the plates were washed with TBS containing 0.05% Tween-20. Forty nanomoles per liter of biotinylated link protein (50 µl) were first allowed to mix with each culture medium (50 µl) and then added as a complex to an antilink protein antibody-coated well. Detection of bound biotinylated link protein was performed with avidin-peroxidase (2 µg/ml). The substrate was tetramethylbenzidine in acetate citrate buffer, pH 6.0. The inter- and intraassay coefficients of variation were 10.2% (n = 10) and 9.3% (n = 10), respectively. In the present experimental conditions, the lowest detectable level of link protein was 10 pmol/liter. The purified link protein was used for the assay standard. Fresh medium supplemented with 10% FBS was used as a negative control.
Statistical analysis
All statistical analysis was performed using StatView
for Macintosh. The Mann-Whitney U test was used for the comparisons
between different groups. P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Examination of Coomassie blue-stained
gels containing unreduced preparations revealed one band with an
apparent molecular mass of 45 kDa (Fig. 1A, right panel).
This protein corresponds in electrophoretic mobility to link protein
from bovine nasal cartilage. Additional characterization of the
cartilage link protein was performed using Western blotting studies.
Western blot of the sample of purified link protein from bovine nasal
cartilage after electrophoretic transfer and incubation with the
antilink protein antibodies revealed a single band at 45 kDa (Fig. 1A, left panel, lane 4).
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). The antilink protein antibodies
detect a single band of approximately 42 kDa, which seems to correspond
to the link protein monomer, in the extracts from whole ovaries [rat
adult cyclic ovary (lane 1) and rat PMSG-treated hCG-stimulated ovary
(lane 2)] and COC lysates (lane 3). No bands were detected in either
the extracts from ovaries or COC lysates when identically processed
preimmune rabbit IgG was used in Western blot analysis (data not
shown).
Culture of rat COCs in serum-free medium or treatment of the cells with
various mucopolysaccharide degradation enzymes showed that antilink
protein antibody reactivity was reduced after elimination of FBS (Fig. 1B, -FCS, lane 2) or digestion with hyaluronidase (HAase, lane 3). The
concentration of link protein was quantitated by measuring the density
of the bands, and the ratio of the reactivity was statistically
analyzed from three independent experiments (Fig. 1C). The Western blot
showed much lower relative link protein concentrations of -FCS and
HAase than of +FCS. The link protein concentrations of -FCS and HAase
were 32.7 ± 8.0% and 18.1 ± 11.7% of that of +FCS,
respectively. Treatment of the purified link protein with
Streptomyces hyaluronidase did not lead to an increase in
the mobility of this band, indicating that link protein itself is
insensitive to hyaluronidase (Kobayashi, H., et al.,
unpublished data, 1998). In addition, treatment of the cells with
keratanase, heparinase, or heparitinase did not affect antibody binding
(data not shown).
As the presence of link protein in rat ovary and COCs in culture was
confirmed by Western blotting, we examined immunohistochemically
distribution and localization of link protein in correlation with COC
expansion in stimulated ovaries. As shown in Fig. 2, intense and diffuse link protein
staining was found in most of the oocytes in ovaries taken from
immature rats before PMSG treatment (Fig. 2A). There was no detectable
reaction product in thecal cells, and the granulosa cells of small
immature follicles (primary and secondary follicles) were completely
devoid of immunoreactivity. No immunostaining was observed when the
primary antibody was substituted with nonimmune IgG (Fig. 2B).
Treatment of the section with hyaluronidase resulted in abolishment of
almost all the immunostaining of the follicular compartment for link
protein (Fig. 2C).
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, D and
D'). The antilink protein antibodies also reacted positively with mural
granulosa cells and to a lesser degree with follicular fluid in mature
follicles that were morphologically at a stage of COC expansion. No
immunostaining was observed when the primary antibody was omitted or
substituted with nonimmune IgG (Fig. 2, E and E'), suggesting that the
staining was specific for the link protein. Again, the staining
intensity was negated by the treatment with hyaluronidase (Fig. 2, F
and F'), suggesting that the link protein is bound to hyaluronic
acid.
In addition, evidence for the presence of link protein in cumulus cell
cultures was obtained using antilink protein antibodies in
immunocytochemical studies. Immunocytochemical experiments further
established the specificity of the immunolabeling. In an in
vitro culture system in which cumulus cells were cultured in the
presence of oocyte-conditioned medium, FBS, and gonadotropin,
immunohistochemistry revealed that the addition of gonadotropin
enhanced link protein expression in most cumulus cells (Fig. 2G). Link
protein was deposited diffusely in the extracellular matrix and on the
cell membrane. However, the cells expressed a small amount of link
protein on the cell surface without gonadotropin (Fig. 2H).
Gonadotropin-stimulated cumulus cells cultured in the absence of FBS
deposited a small amount of link protein on the cell membrane (data not
shown). Furthermore, when cumulus cells were cultured in the medium
supplemented with FBS and gonadotropin in the absence of
oocyte-conditioned medium, weak reactivity was observed in the
extracellular matrix of cumulus cells (data not shown), suggesting that
an oocyte factor is also required for link protein production from
cumulus cells. The staining of cultured cumulus cells with antilink
protein antibody was completely negated by the addition of
hyaluronidase (Fig. 2I). Experiments using three independent cultured
cumulus cells gave essentially identical results, demonstrating that
gonadotropin-stimulated cells increased the deposition of link protein
into the extracellular matrix compared with that in the control without
the addition of gonadotropin or FBS.
Isolated rat cumulus cells in the presence of oocyte-conditioned medium
were incubated with gonadotropin for 12, 24, and 36 h, and the
amount of link protein in the medium was determined by a specific ELISA
(Fig. 3). ELISA data revealed that
gonadotropin caused a significant increase in cumulus cell link protein
production. These stimulatory effects were seen after as little as
12 h and were prominent after 24 h of incubation. The rate of
secretion of link protein increased approximately 240% by 24 h
(mean ± SD, 20.6 ± 4.4 ng/104
cells·24 h in the presence of gonadotropin vs. 8.6 ±
2.4 ng/104 cells·24 h in the absence of
gonadotropin).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The antilink protein polyclonal antibody produced by us detected a
specific 42-kDa band on a Western blot of the stimulated ovaries and
COC lysate as well as the 45-kDa cartilage link protein. The presence
in cumulus cell extracts of the 42-kDa protein that reacted with the
antilink protein antibody to cartilage link protein indicated that the
42-kDa protein shares an epitope with bovine nasal cartilage link
protein. Extracts of rat COC cultures were found to contain a protein
with molecular mass and electrophoretic behavior similar to those of
bovine nasal cartilage link proteins (20, 21, 24, 31). As previously
reported (24, 32), at least three link proteins (
48, 44, and 40
kDa) were present in normal articular cartilage extracts under
nonreducing conditions, but only one cumulus cell link protein band was
identified in the present study. Although the 42-kDa protein is thought
to be a monomer of link protein, the reason for this difference is as
yet unknown. Neame et al. (33) reported that rat
chondrosarcoma-derived link protein has a core mass of 38,564 plus one
N-linked oligosaccharide of 2,500. This is very close to the
42-kDa protein reported in the present study and significantly lower
than the molecular mass of bovine link protein.
In addition, we have identified the immunoreactive link protein in rat ovaries during the preovulatory period that were morphologically at a stage of COC expansion and in COC cultures or cumulus cell cultures supplemented with oocyte-conditioned medium, using immunolocalization with a specific antilink protein antibody. With the immunohistochemical approach, it is possible to determine the specific tissue compartments that express link protein antigen during follicular development. The results of this study showed a periovulatory increase in cumulus cell and mural granulosa cell link protein staining of gonadotropin-treated animals.
ELISA analysis in cumulus cell cultures also indicated a synthesis of immunoreactive link protein. We demonstrated that gonadotropin significantly stimulates cumulus cell link protein production in an in vitro culture system. Protein with characteristics similar to those of the bovine nasal cartilage link protein was identified in the cumulus cell culture medium, indicating that it is exported from the cells in the absence of FBS.
The presence and synthesis of link protein in cumulus cell cultures are
of importance because this is the first time, to our knowledge, that
immunoreactive link protein has been definitively demonstrated in
cumulus cells. The cartilage and noncartilage link proteins and
proteoglycan core protein are reported to induce stabilization between
hyaluronic acid and also bind to each other (12, 16, 17, 18, 19). It is
possible that link protein is involved in the stabilization of
hyaluronic acid-rich aggregates. It would appear that the link protein
is involved in interaction with proteins of the inter--trypsin
inhibitor family or other extracellular matrix proteins. The important
findings are that cultured cumulus cells deposited link protein mainly
into the extracellular matrix and that link protein is released from
the tissues and cultured cumulus cells by hyaluronidase digestion,
which suggest that link protein is not anchored to the cell membranes
lining the extracellular space and is bound to hyaluronic acid. Link
protein itself is not sensitive to hyaluronidase (Kobayashi, H.,
et al., unpublished data, 1998). This suggests that the
release was dependent on the degradation of hyaluronic acid, rather
than being brought about by proteolytic activity. The interaction among
hyaluronic acid, link protein, and extracellular matrix proteins,
including inter--trypsin inhibitor and dermatan sulfate
proteoglycan, on the cumulus cell surface may induce and strengthen COC
expansion and lead to ovarian follicle maturation.
We also demonstrated that immunoreactivity for link protein has been localized primarily to oocyte regardless of the animal age and hormonal condition. It is possible that oocyte-derived link protein functions to stabilize an aggregate structure of the hyaluronic acid-rich matrix on the oocyte. Alternatively, link protein may have a function other than the stabilization of proteoglycan aggregates. Although link protein is abundantly expressed in cartilage (34), the tissue distribution of the link protein is not limited to the cartilaginous tissue, and expression of link protein messenger RNA has been found clearly in brain, aorta, embryonic retina, lens epithelium, and mesonephros by Northern blot analysis (35, 36). These results suggest that the role of the link protein-producing cells may be related to the sustaining supply or transportation of link protein to extracellular matrix. However, the exact role of link protein in the oocyte is as yet unknown.
In conclusion, we describe the presence and synthesis of immunoreactive
link protein in stimulated rat ovary. Gonadotropin increases link
protein production in cumulus cell cultures. It was established that
cumulus cells significantly produce hyaluronic acid in the presence of
appropriate gonadotropin (37) and FBS and that inter--trypsin
inhibitor is accumulated in follicular fluids from plasma through
increased vascular permeability just before ovulation (9). The link
protein may stabilize the interaction between hyaluronic acid and
extracellular matrix proteins, such as inter--trypsin inhibitor on
cumulus cells, by triggering COC expansion and may function as a part
of this complex pathway. These results provide an intriguing
area of investigation regarding the molecular mechanism of the
hyaluronic acid-link protein-inter--trypsin inhibitor system in
ovarian follicle development. Further work is needed to characterize
cumulus cell-associated link protein, to clarify possible relationships
between COC link protein and cartilage link proteins, and to determine
its function in COC expansion. The distribution of link protein by
in situ hybridization during different stages of ovarian
follicle development is underway.
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Acknowledgments |
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Received January 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or
without (•) gonadotropin. Concentrations of link protein in culture
medium were measured as described in Materials and
Methods. Values represent the mean ± SD of
one experiment. *, P < 0.05. Experiments using
four independent cultured cumulus cells gave essentially identical
results or showed similar tendencies.