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Reproductive Endocrinology |
6ß11
Department of Gynecology and Obstetrics, Faculty of Medicine (H.F., T.H., K.N., S.Y., T.M.), Institute for Virus Research (M.U.), and Chest Disease Research Institute (M.M.), Kyoto University, Sakyo-ku, Kyoto 606–01, Japan
Address all correspondence and requests for reprints to: Hiroshi Fujiwara, M.D., Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606–01, Japan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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6, and that human GC express integrin
6ß1, but not
6ß4.
In the present study, we examined the expression of laminin (LN), the
ligand for integrin
6ß1. Flow cytometry
showed that LN was bound to the cell surface of some GC obtained from
preovulatory follicles of patients undergoing in vitro
fertilization. Immunohistochemistry showed that LN was detected between
luteinizing GC in the early corpora lutea.
To examine the effect of LN on steroidogenesis by human luteinizing GC, GC obtained from patients undergoing in vitro fertilization were cultured on mouse LN-coated or noncoated plastic dishes in medium containing 5% FCS for 24 h. In the absence or presence of hCG (1 IU/mL), GC cultured on LN-coated dishes produced 0.70- and 0.67-fold less progesterone than those on noncoated dishes, respectively (P < 0.05).
We examined the effect of the interaction of integrin
6ß1 and LN on
steroidogenesis by human luteinizing GC. We cultured GC with 5 µg/mL
of the anti-6 mAb GoH3, which inhibits the
interaction between human integrin
6ß1 and mouse LN, or
with a control rat mAb (TER199) on mouse LN-coated dishes in serum-free
medium for 24 h. In the absence or presence of hCG (1 IU/mL), GC
cultured with GoH3 produced 1.97- and 1.94-fold more progesterone than
the control cells (P < 0.01 and
P < 0.05, respectively). In contrast, when GC were
cultured on dishes coated with type IV collagen, progesterone
production was not enhanced by GoH3. Furthermore, the
anti-6 mAb OG-1, which does not inhibit the
interaction between integrin 6ß1
and LN, had no effect on the progesterone production by GC cultured on
LN.
These results indicate that LN suppresses the luteinization of human
luteinizing GC via integrin
6ß1 and that integrin
6ß1 regulates the
luteinization of human GC during the periovulatory phase.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We raised the mAb OG-1, which recognizes a cell surface molecule of
human granulosa cells (GC) (7). N-Terminal amino acid sequencing of the
purified OG-1 antigen showed that it is identical to human integrin
6 (8). We also raised the mAb POG-2 that
recognizes a cell surface molecule of the porcine GC and demonstrated
that POG-2 antigen is a porcine homolog of integrin
6 by N-terminal amino acid sequencing (9). The
expression profiles of integrin 6 differ
between species. In the human ovary, integrin
6 is expressed on GC of medium to large
follicles and on luteinizing GC in the early luteal phase. The
expression level was highest during the periovulatory stage. In
contrast, in the porcine ovary, integrin 6 was
expressed on GC in the small follicles (1–2 mm in diameter) with
maximal immunoreactivity and was undetectable in corpora lutea (CL).
These stage-specific expression profiles suggested the involvement of
integrin 6 in ovarian physiology (10).
We reported that human GC express integrin ß1,
but not integrin ß4, indicating that integrin
6 expressed on GC forms a heterodimer with
ß1, but not with ß4 (8). As the
ligand for integrin
6ß1 is laminin (LN)
(11), we examined the expression of LN on GC and the physiological
function of the interaction between integrin
6ß1 and LN.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine antihuman LN mAb, LAM89 (IgG1 isotype), was purchased
from Sigma Chemical Co. (St. Louis, MO). The murine antihuman integrin
6 mAb OG-1 (IgG1) was raised in our laboratory
(7). The murine antitrinitrophenyl (anti-TNP) mAb (IgG1) was used as a
control (12). The rat antimouse integrin 6
mAb, GoH3 (IgG2a), which cross-reacts with human integrin
6 (13, 14), was purchased from Serotec
(Oxford, UK). This mAb partially blocks the interaction of integrin
6 and LN (14). The rat antimouse erythroid
cell mAb, TER199 (IgG2a), a gift from Dr. T. Kina (Chest Disease
Research Institute, Kyoto University, Kyoto, Japan), was used as a
control (15).
The mAb GoH3 inhibits the interaction between human integrin
6ß1 and murine LN, but
OG-1 does not, as shown below.
Human ovaries
Preovulatory growing ovarian follicles (n = 4) and CL of the early luteal phase (n = 6) were obtained from 10 patients, aged 33–45 yr, with regular menstrual cycles, undergoing surgery for benign disease. Written informed consent was obtained from each patient.
Follicles were morphologically evaluated by staining cryosections with hematoxylin and eosin (HE). Follicles obtained in the follicular phase (18–20 mm in diameter) with GC having mitotic figures and regularly shaped nuclei, cytoplasm, and stratified layers were classified as preovulatory growing follicles (16).
The postovulatory date of CL was evaluated according to the histological dating described by Corner (17), using HE-stained tissue sections of 10% formalin-fixed and paraffin-embedded samples. In this work, the term CL day was used according to his definition. For example, CL day 2 is the next day of ovulation.
Indirect immunohistochemical staining of frozen sections
Indirect immunofluorescence histochemistry proceeded as previously described (7). Each specimen was embedded in OCT compound (Tissue-Tec, Miles Scientific, Naperville, IL), snap-frozen in liquid nitrogen, and stored at -80 C. Frozen tissues were sliced to 7-µm thickness using a cryostat microtome (Cryocut 1800, Reichert-Jung, Heidelberg, Germany), immediately air-dried on Neoplene (Nisshin EM, Tokyo, Japan)-coated glass slides, and fixed in acetone at -20 C for 5 min. The slides were incubated with the anti-LN mAb (ascites diluted 1:1000), OG-1 (5 µg/mL), or the anti-TNP mAb (5 µg/mL; negative control) for 40 min at room temperature. After washing in phosphate-buffered saline (PBS), they were incubated with the fluorescein isothiocyanate-conjugated rabbit antimouse Ig antibody (diluted 1:40; Dakopatts, Glostrup, Denmark) for 40 min at room temperature in the dark. The slides were washed, mounted with Perma Fluor Aqueous Mounting Medium (Immunon, Pittsburgh, PA), and examined under a fluorescence microscope (Nikon, Tokyo, Japan). Serial cryosections were also stained with HE after acetone fixation.
Isolation of human luteinizing GC
Human GC were isolated from 26 patients, aged 28–38 yr, undergoing in vitro fertilization (IVF), as previously reported (18). Briefly, patients receiving a GnRH analog (buserelin acetate, Hoechst Japan Co., Tokyo, Japan) beginning on the first day of the cycle were hyperstimulated with human menopausal gonadotropin (Organon Japan Co., Tokyo, Japan) until the follicles reached maturity. Follicles were aspirated 36 h after the administration of hCG (Mochida Pharmaceutical Co., Osaka, Japan). The follicular fluid was centrifuged, and the resuspended GC were overlaid on Ficoll-Hypaque and centrifuged at 400 x g for 30 min. Cells were collected from the interphase.
Flow cytometric analysis of LN bound to isolated human luteinizing GC
The isolated human GC were washed in Hanks’ balanced salt solution (HBSS) with 0.1% BSA and 0.1% NaN3, sedimented by centrifugation, and incubated with 5 µL anti-LN mAb (ascites diluted 1:20) or the anti-TNP mAb (100 µg/mL) for 30 min at 4 C. After washing in HBSS, the cell pellet was incubated with fluorescein isothiocyanate-conjugated rabbit antimouse Ig for 30 min at 4 C in the dark. After washing in HBSS, the cells were resuspended in the same solution, and viable cells were analyzed by flow cytometry (FACScan, Becton Dickinson Immunocytometry Systems Japan, Tokyo, Japan). The ratio of contaminating monocytes, identified by the anti-CD14 mAb (Becton Dickinson), was less than 3%.
Culture of human luteinizing GC on LN-coated or noncoated dishes in medium containing 5% FCS
Dishes were coated as previously described with minor modification (14). Briefly, mouse LN (Becton Dickinson Labware, Bedford, MA), composed of the whole LN fragments and with a molecular mass of 900,000 daltons, was dissolved at 40 µg/mL in PBS, and 100 µL were placed in each well (6.3 µg/cm2 LN) of 96-well noncoated polystyrene dishes (Corning Glass Works, Corning, NY). The dishes were stored at 4 C overnight and washed in PBS before use. The GC isolated as described above were suspended in DMEM-Ham’s F-12 medium (1:1, vol/vol; Life Technologies, Gaithersburg, MD) medium containing 5% FCS (Flow Laboratories, McLean, VA) and 10 mmol/L HEPES (Nacalai Tesque, Kyoto, Japan) without antibiotics at a density of 3 x 105 cells/mL. The cells were inoculated on 96-well noncoated polystyrene or on mouse LN-coated dishes at 100 µL/well and cultured in the absence or presence of hCG (1 IU/mL; Serono Japan, Tokyo, Japan). After 24 h, the supernatant was collected to assay steroid production. To assay estradiol, 10-7 mol/L testosterone was added to the medium. The cells were detached by incubation with PBS containing with 0.05% trypsin (Difco, Detroit, MI) and 0.05% ethylenediamine tetraacetate (Nacalai Tesque), then the number of viable GC per well were counted under microscopy by trypan blue exclusion.
Inhibition assay of BeWo cell attachment to LN by the mAbs OG-1 and GoH3
BeWo, a human choriocarcinoma cell line, was obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). Single cells obtained with trypsin treatment were analyzed by flow cytometry as described above, using OG-1 mAb and the mouse antihuman integrin ß1 mAb (P4C10, Life Technologies). For assay of the inhibition of attachment, single cells were incubated with the mAb OG-1 (0, 10, and 20 µg/mL), the mAb GoH3 (0, 2.5, and 5 µg/mL), or control mouse (anti-TNP; 20 µg/mL) and rat (TER199; 5 µg/mL) mAbs at 4 C for 30 min. The cells were inoculated on commercially available dishes coated with mouse LN (Becton Dickinson Labware), in serum-free DMEM-Ham’s F-12 medium at a density of 2 x 105 cells/mL. After 6 h, the wells were gently rinsed three times with PBS to remove unattached cells, then the number of the attached viable cells was counted as described above.
Culture of human luteinizing GC with the antiintegrin
6 mAbs on LN- or type IV collagen
(CLN)-coated dishes in serum-free medium
The isolated GC were washed twice in serum-free DMEM-Ham’s F-12
medium with 10 mmol/L HEPES without antibiotics. The cells were
suspended at a density of 3 x 105 cells/mL in
the same serum-free medium, then incubated with the antiintegrin
6 mAb (GoH3 or OG-1) or the control mAb
(TER199 or anti-TNP, respectively) at a final concentration of 5
µg/mL at 4 C for 30 min. The cells were inoculated on 96-well mouse
LN-coated or mouse type IV CLN-coated dishes (Becton Dickinson Labware;
100 µL/well) in the absence or presence of hCG (1 IU/mL). After
24 h, the supernatants were collected to assay steroid production,
and the number of viable GC per well was counted as described
above.
Assay of steroid production by human luteinizing GC
The concentrations of progesterone and estradiol were measured using RIA kits (Daiichi Radio Isotope Research, Tokyo, Japan). Inter- and intraassay coefficients of variation were 6.5% and 5.3% for the progesterone assay and 7.4% and 6.3% for the estradiol assay, respectively.
Statistics
Each experiment was performed in triplicate. Data are shown as the mean \ SEM. The inhibition assay of cell attachment was examined by one-way ANOVA, followed by Scheffe’s F test. Steroid production was analyzed by two-tailed paired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In preovulatory follicles (18–20 mm in diameter; n = 4), LN
was detected on the basal lamina, around vessels, and in stroma,
including theca interna cells, but not between GC (Fig. 1B
). In CL of the early luteal phase (CL day 2, n =
2; day 4, n = 2; day 5, n = 2), LN was expressed between
luteinizing GC from the next day of ovulation (Fig. 2B).
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The percentage of LN-bound human luteinizing GC varied greatly
between individuals, ranging from undetectable (0%) to 51.2% (n
= 12; mean \ SD, 14.5 \ 14.4%). LN was
detected on the cell surface region of GC (Fig. 3). A
representative histogram is shown in Fig. 4.
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After 24-h culture, there was no difference in cell morphology or
in the number of viable human luteinizing GC between the groups
cultured on LN-coated or noncoated dishes. In the absence or presence
of hCG, progesterone production by cells cultured on LN-coated dishes
was 0.70- and 0.67-fold less than that in cells on noncoated dishes
(P = 0.0123 and P = 0.0233,
respectively; Fig. 5A). There was no significant
difference in estradiol production (Fig. 5B).
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Flow cytometry showed that integrins 6
and ß1 are expressed on 75 \ 5.3% and
79 \ 6.2% of BeWo cells, respectively (n = 3). A histogram
is shown in Fig. 6.
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6 mAbs on
progesterone and estradiol production by human luteinizing GC cultured
on LN-coated or type IV CLN-coated dishes
Human luteinizing GC were cultured with the antiintegrin
6 mAb GoH3 or with the control mAb on mouse
LN-coated dishes in serum-free medium. After 24 h, there was no
difference in cell morphology or in the number of viable cells between
the two groups. Progesterone production by GC cultured with GoH3
without hCG was 1.97-fold higher (P = 0.0031) than that
by GC cultured with control mAb and was 1.94-fold higher
(P = 0.0177) than the control value when cultured with
1 IU/mL of hCG (Fig. 8A). There was no difference in the
level of estradiol production (data not shown). In contrast, GoH3 did
not enhance progesterone production by GC cultured on type IV
CLN-coated dishes (Fig. 8B) and had no effect on estradiol production
(data not shown).
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6 mAb, and the control group when GC were
cultured on either LN-coated dishes or type IV CLN-coated dishes (data
not shown). There was no difference in estradiol production between the
two groups (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6ß1 is a
differentiation-related cell surface molecule of human GC and is
expressed at the highest levels during the periovulatory phase (7, 8).
In this study, we examined the expression of LN, the ligand for
integrin 6ß1, during
the periovulatory phase. Although immunoreactive LN was undetectable
between GC in preovulatory follicles, LN was detected between
luteinizing GC by immunohistochemistry after ovulation. Moreover, LN
was detected by flow cytometry on the cell surface of luteinizing GC
obtained from the patients undergoing IVF, indicating that LN is
actually bound to GC in vivo before ovulation. These
findings demonstrated that the expression of LN on GC increases during
ovulation and suggest a physiological interaction between integrin
6ß1 and LN, from the
preovulatory to the early luteal phase. We examined the effect of LN on steroidogenesis by human luteinizing GC. When cultured on mouse LN-coated dishes for 24 h, human luteinizing GC produced less progesterone than those on noncoated dishes. As there were no differences in estradiol production or cell numbers between the two groups, this result indicates that LN suppressed luteinization of GC without decreasing cell viability.
An inhibition assay of BeWo cell attachment showed that the interaction
between human integrin
6ß1 and mouse LN was
inhibited by the antihuman and antimouse integrin
6 mAb GoH3, but not by our antihuman integrin
6 mAb OG-1. Using the blocking mAb (GoH3) and
the nonblocking mAb (OG-1), we examined the effect of the interaction
between integrin 6ß1
and LN on the luteinization of human luteinizing GC. GoH3 enhanced
progesterone production by human luteinizing GC cultured on LN-coated
dishes without altering cell viability. In contrast, it did not enhance
progesterone production by those cells cultured on dishes coated with
type IV CLN, which is not the ligand for integrin
6ß1. These results
demonstrate that progesterone production was enhanced by specific
blocking between integrin
6ß1 and LN.
Furthermore, the nonblocking antiintegrin 6
mAb, OG-1, had no effect on progesterone production by GC cultured on
LN-coated dishes. This result indicates that binding to integrin
6ß1 by the mAb does
not affect progesterone production by GC cultured on LN and supported
the idea that the specific blocking of the interaction between integrin
6ß1 and LN enhances
it. Therefore, we concluded that LN suppresses GC luteinization via
interaction with integrin
6ß1.
GC luteinization is a process in which the capacity of progesterone
production is augmented, coupled with the morphological changes in
cytoplasmic organelles. Human GC gradually differentiate to large
luteal cells within 5 days after ovulation (17). The serum level of
progesterone gradually increases until it reaches a peak 5–6 days
after LH surge (19). We reported that the expression of several
differentiation-related cell surface molecules on luteinizing GC, such
as dipeptidyl peptidase IV, leukocyte functional antigen-3, HLA-DR, and
human CL antigen-1, increases during CL formation (2, 3, 4, 20). We
consider that the culture of human luteinizing GC obtained from
patients undergoing IVF is a model of the stage of CL formation. Using
this culture system, we examined the effects of hCG, interleukin-1
(IL-1), and tumor necrosis factor- (TNF) on the induction of
differentiation-related molecules on cultured GC. The expression of
dipeptidyl peptidase IV was enhanced by TNF and IL-1, but not by
hCG, and that of leukocyte functional antigen-3 was enhanced by
TNF-, but not by hCG (4, 18). These findings showed that TNF and
IL-1 induce luteal cell differentiation. On the other hand, hCG
suppressed the expression of human CL antigen-1 antigen, whereas its
expression on luteinizing GC increased during CL formation in
vivo and in vitro (20). These findings suggest that
after ovulation, a local regulatory mechanism as well as an endocrine
system operate in the differentiation process of human GC into luteal
cells. In this study, we demonstrated that LN suppresses the
luteinization of human GC via interaction with integrin
6ß1, indicating that the integrin
6ß1-LN interaction
serves as a local inhibitory regulator of luteinization. This study is
the first to show that integrins regulate ovarian cell differentiation.
It is reasonable that the intensity of integrin
6ß1 expression on
human luteinizing GC decreases from the early luteal to the midluteal
phase (7, 8), as human GC become fully luteinize toward the midluteal
phase. Amsterdam et al. reported that the luteinization of
human luteinizing GC is enhanced when cultured on mixed bovine
extracellular matrices (ECM) composed of CLN (types I, III, and IV),
heparin sulfate proteoglycans, LN, entactin, elastin, fibronectin,
dermatan sulfate, and chondroitin sulfate proteoglycans (21). Recently,
they also reported that several ECM have differential effects on
apoptosis and steroidogenesis of cultured rat mature GC, showing that
LN suppressed progesterone production by rat mature GC (22). Taken
together with our findings, it is strongly suggested that each ECM has
a differential regulatory effect on luteinization of human GC.
As blocking of the interaction between integrin
6ß1 and LN enhanced
progesterone production independently of hCG, a system(s) other than
that of the LH and LH receptor may operate in the inhibitory effect of
the integrin 6ß1-LN
interaction on the luteinization of human GC. Several systems,
including the focal adhesion kinase (pp125), are involved in the
intracellular signaling via integrins (23). Therefore, approaches using
integrins may help clarify the molecular mechanisms of luteinization of
human GC.
Leardkamolkarn et al. showed by immunoelectron microscopy
that rat GC produce LN (24), and Zhao Y. et al. proved by
Northern blotting that bovine GC produce LN-B2 chain (25). Similarly,
human GC may secrete LN in the follicular fluid, as flow cytometry
detected LN on the cell surface of preovulatory GC. During the
menstrual cycle of women, serum progesterone rapidly increases just
before the LH surge (26). The increase in serum progesterone is
considered to be important for the onset of the LH surge (27). In the
IVF program, serum progesterone occasionally increases before hCG
administration even when the endogenous LH surge is suppressed by a
GnRH analog (28). These progesterone increases are considered to be due
to the luteinization of GC before the LH surge, but the precise
mechanism of its regulation is unknown. If integrin
6ß1 expressed on GC in
large follicles interacts with LN and suppresses the luteinization of
GC, this interaction may regulate the timing of the onset of the LH
surge or prevent GC from premature luteinization, which interferes with
oocyte maturation (29, 30).
In conclusion, this study suggests a physiological interaction between
GC and LN from the preovulatory to the early luteal phase and that LN
suppresses GC luteinization. We demonstrated that the integrin
6ß1-LN interaction
suppresses the luteinization of human GC and proposed that integrin
6ß1 is a local
regulator of GC luteinization during the periovulatory phase. These
findings support the idea that a differentiation stage-specifically
expressed molecule(s) on ovarian cells is involved in their
differentiation and function.
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Acknowledgments |
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Footnotes |
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Received August 27, 1996.
Revised February 5, 1997.
Revised April 1, 1997.
Accepted April 11, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6 is a differentiation antigen of human granulosa cells. J Clin Endocrinol Metab. 80:2899–2905.6 is a differentiation
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) functions of
murine B cells. J Immunol. 144:4571–4578.[Abstract]
6ß1 but not 6ß4 functions as major laminin receptor for
fragment E8. J Cell Biol. 110:2145–2155.This article has been cited by other articles:
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S. K. Saha, P. Ghosh, A. Konar, S. Bhattacharya, and S. S. Roy Differential Expression of Procollagen Lysine 2-Oxoglutarate 5-Deoxygenase and Matrix Metalloproteinase Isoforms in Hypothyroid Rat Ovary and Disintegration of Extracellular Matrix Endocrinology, July 1, 2005; 146(7): 2963 - 2975. [Abstract] [Full Text] [PDF]
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M. Egawa, S. Yoshioka, T. Higuchi, Y. Sato, K. Tatsumi, H. Fujiwara, and S. Fujii Ephrin B1 Is Expressed on Human Luteinizing Granulosa Cells in Corpora Lutea of the Early Luteal Phase: The Possible Involvement of the B Class Eph-Ephrin System during Corpus Luteum Formation J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4384 - 4392. [Abstract] [Full Text] [PDF]
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K. Kosaka, H. Fujiwara, K. Tatsumi, S. Yoshioka, Y. Sato, H. Egawa, T. Higuchi, T. Nakayama, M. Ueda, M. Maeda, et al. Human Chorionic Gonadotropin (HCG) Activates Monocytes to Produce Interleukin-8 via a Different Pathway from Luteinizing Hormone/HCG Receptor System J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5199 - 5208. [Abstract] [Full Text] [PDF]
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K. R. Park, T. Inoue, M. Ueda, T. Hirano, T. Higuchi, M. Maeda, I. Konishi, H. Fujiwara, and S. Fujii CD9 is expressed on human endometrial epithelial cells in association with integrins {alpha}6, {alpha}3 and {beta}1 Mol. Hum. Reprod., March 1, 2000; 6(3): 252 - 257. [Abstract] [Full Text] [PDF]
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T. Higuchi, H. Fujiwara, S. Yamada, K. Tatsumi, N. Kataoka, K. Itoh, M. Maeda, J. Fujita, and S. Fujii Co-expression of integrin-associated protein (IAP/CD47) and its ligand thrombospondin-1 on human granulosa and large luteal cells Mol. Hum. Reprod., October 1, 1999; 5(10): 920 - 926. [Abstract] [Full Text] [PDF]
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S. Yamada, H. Fujiwara, T. Honda, T. Higuchi, T. Nakayama, T. Inoue, M. Maeda, and S. Fujii Human granulosa cells express integrin {alpha}2 and collagen type IV: possible involvement of collagen type IV in granulosa cell luteinization Mol. Hum. Reprod., July 1, 1999; 5(7): 607 - 617. [Abstract] [Full Text] [PDF]
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Y. Takao, H. Fujiwara, S. Yamada, T. Hirano, M. Maeda, S. Fujii, and M. Ueda CD9 is expressed on the cell surface of human granulosa cells and associated with integrin {alpha}6ß1 Mol. Hum. Reprod., April 1, 1999; 5(4): 303 - 310. [Abstract] [Full Text] [PDF]
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