Molecular Human Reproduction, Vol. 5, No. 7, 607-617,
July 1999
© 1999 European Society of Human Reproduction and Embryology
Human granulosa cells express integrin 
2 and collagen type IV: possible involvement of collagen type IV in granulosa cell luteinization
1 Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, 2 Department of Gynecology and Obstetrics, Kurashiki Central Hospital, Kurashiki, 710-8602, and 3 Institute for Frontier Medical Science, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
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Previously, it has been shown that integrin
6ß1 expressed on human granulosa cells regulates luteinization in co-operation with its ligand, laminin. In this study, integrin 2 was immunohistochemically demonstrated to be expressed on granulosa and large luteal cells. It was also detected on luteinizing theca interna cells after ovulation. Immunoreactive collagen type IV, which is one of the ligands for integrin 2ß1, was detected around granulosa cells in the pre-ovulatory follicles and its expression was rapidly increased during ovulation. By flow cytometry, collagen type IV was detected on the cell surface of luteinizing granulosa cells isolated from pre-ovulatory follicles, confirming the physiological interaction between granulosa cells and collagen type IV. Collagen type IV in follicular fluid was positively related with progesterone concentration. In 4-day cultures of granulosa cells, collagen type IV in the media was significantly increased by human chorionic gonadotrophin (HCG). The progesterone production was significantly attenuated when granulosa cells were cultured on collagen type IV-coated dishes, suggesting that collagen type IV suppresses granulosa cell luteinization. These findings show that collagen type IV, a ligand for integrin 2ß1, is rapidly produced around luteinizing granulosa cells during ovulation, probably under the control of luteinizing hormone (LH) and suggest that collagen type IV is a new parameter and/or regulator of granulosa cell luteinization in the periovulatory phases.
collagen Type IV/granulosa cells/integrin 
2/luteal cells/luteinization
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Introduction |
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To investigate the regulatory factors involved in ovarian physiology, we raised monoclonal antibodies (mAb) against human or porcine ovarian cells (Fujiwara et al., 1992a
,b, 1993a,Fujiwara et al., b, 1995; Hattori et al., 1995). Of these mAb, OG-1 and POG-2 recognize cell surface molecules of human and porcine granulosa cells respectively (Fujiwara et al., 1993b, 1995). N-terminal amino acid sequencing of the purified OG-1 and POG-2 antigens revealed that they are identical to integrin 6 (Fujiwara et al., 1995; Honda et al., 1995). In the human ovary, integrin 6ß1 was expressed on the cell surface of luteinizing granulosa cells in the early luteal phase and its ligand, laminin, was also detected around these granulosa cells. Further examination revealed that laminin suppressed progesterone production via the interaction with integrin 6ß1 (Fujiwara et al., 1997).
Integrins are heterodimeric molecules on the cell surface, consisting of and ß chains (Hynes, 1992). They mediate the interaction between the cells and extracellular matrices. It is reported that the interaction with extracellular matrices via integrins evoke intracellular signal transduction (Shaller and Parsons, 1994). Recently, we have shown that integrin 5 is expressed on human luteinizing granulosa cells. The ligand for integrin 5ß1, fibronectin, is also produced by luteinizing granulosa cells and its production rapidly increases during ovulation, suggesting the involvement of extracellular matrices in granulosa cell luteinization.
In this study, we immunohistochemically demonstrated that integrin 2 was expressed on human granulosa cells in both pre- and post-ovulatory follicles and that collagen type IV, one of the ligands for integrin 2ß1 (Staatz et al., 1989), was detected among granulosa cells in these follicles. Since the production of collagen type IV was observed to increase promptly during luteinization of granulosa cells, we examined the concentration of collagen type IV in follicular fluid in comparison with progesterone and oestradiol concentrations. To elucidate the physiological role of collagen type IV in the granulosa cell luteinization, we also examined the effect of collagen type IV on progesterone production by luteinizing granulosa cells.
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Antibodies
The murine anti-human collagen type IV mAb, 4H12 [immunoglobulin (Ig)G1 isotype], was obtained from Fuji Chemical Industries Ltd, (Toyama, Japan). The murine anti-human integrin
2 mAb, P1E6 (IgG1 isotype) and the murine anti-integrin ß1 mAb, P4C10 (IgG1 isotype) were purchased from Gibco BRL (Gaithersburg, MD, USA) (Wayner et al., 1988; Carter et al., 1990). The murine anti-trinitrophenyl (TNP) mAb (IgG1) was used as a control (Tsujimura et al., 1990).
Human ovaries
The growing follicles (3–12 mm in diameter, n = 6), pre-ovulatory follicles (18–20 mm in diameter, n = 4), corpora lutea (CL) of the menstrual cycle (n = 21) were obtained from 31 women, aged 21–46 years. They had undergone unilateral ovarian cystectomy or oophorectomy and contralateral wedge resection to treat benign ovarian tumours. All women had a history of regular menstrual cycles (28–30 days) and their ovulatory basal body temperature charts were of normal luteal phase length. CL of pregnancy were obtained from five pregnant patients aged 28–46 years, who had undergone hysterectomy at 6, 7, 9, 11, or 14 weeks gestation due to uterine myoma and/or cervical cancer. In all five patients, fetal growth was normal on ultrasonographic examination. The gestational weeks were determined from the last menstrual period in the patient. Macroscopically- and microscopically-normal regions of these tissues were used for this study. Written informed consent was obtained from each patient prior to the study.
Follicles were morphologically evaluated by staining cryosections with haematoxylin and eosin. Follicles obtained in the follicular phase with granulosa cells having mitotic figures and regularly shaped nuclei, cytoplasm, and stratified layers were classified as growing follicles (Ryan, 1981). If the judgement is difficult, the haematoxylin and eosin stained sections from the identical samples that were fixed with 10% formalin and embedded with paraffin were used.
The post-ovulatory date of CL was evaluated according to the histological dating described by Corner GW, using haematoxylin and eosin stained tissue sections of 10% formalin-fixed and paraffin-embedded samples (Corner, 1956). In this work, the term corpus luteum (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 was carried out as previously described (Honda et al., 1995). Each specimen was embedded in OCT compound (Tissue-Tec, Miles Scientific, Naperville, IL, USA), 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 anti-integrin 2ß1, P1E6 (ascites diluted 1:1000), anti-collagen type IV mAb (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 (FITC)-conjugated rabbit anti-mouse immunoglobulin (Ig) antibody (diluted 1:40; Dakopatts A/S, 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, USA), and examined under a fluorescence microscope (Nikon, Tokyo, Japan). Serial cryosections were also stained with haematoxylin and eosin after acetone fixation. The intensity of antigen expression was graded from – to ++ based on its fluorescence intensity (–, absence of staining; +, weak staining; ++, intense staining).
Isolation of human luteinizing granulosa cells
Human granulosa cells were isolated from 26 patients, aged 28–38 years, undergoing in-vitro fertilization (IVF) treatment, as reported (Fujiwara et al., 1994). Briefly, patients receiving a gonadotrophin-releasing hormone (GnRH) analogue (buserelin acetate; Hoechst Marion Roussel Ltd, Tokyo, Japan), beginning on the first day of the cycle, were stimulated with human menopausal gonadotrophin (HMG; Organon Japan Co Ltd, Tokyo, Japan) until the follicles reached maturity. Follicles were aspirated 36 h after the administration of human chorionic gonadotrophin (HCG; Mochida Pharmaceutical Co Ltd, Osaka, Japan). The follicular fluid was centrifuged, and the resuspended granulosa cells were overlayered on Ficoll–Hypaque and centrifuged at 400 g for 30 min. The cells were collected from the interface.
Flow cytometrical analysis of cell surface expression of integrins 2, ß1 and collagen type IV on isolated human luteinizing granulosa cells
The isolated human granulosa cells were washed in Hanks' balanced salt solution (HBSS) with 0.1% bovine serum albumin and 0.1% NaN3, sedimented by centrifugation and incubated with 5 µl of anti-integrin 2 mAb (ascites diluted 1:20), anti-integrin ß1 mAb (ascites diluted 1:20), anti-collagen type IV mAb (100 µg/ml) or the anti-TNP mAb (100 µg/ml) for 30 min at 4°C. After washing in HBSS, the cell pellet was incubated with FITC-conjugated rabbit anti-mouse 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 analysed by flow cytometry (FACScan; Becton Dickinson Immunocytometry Systems Japan, Tokyo, Japan). The ratio of contaminating monocytes, identified by the anti-CD14 mAb (Becton Dickinson Labware, Bedford, MA, USA), was <3%. These experiments were repeated five times. In some cases, the cells after the incubation with second antibody were suspended in PBS/glycerin (1:1 v/v) and observed under a fluorescence microscope.
Culture of human luteinizing granulosa cells on collagen type IV-coated or non-coated dishes
The isolated granulosa cells were incubated with collagenase (type 1A, Sigma, St. Louis, MO, USA) at 37°C for 2 h to detach collagen type IV from their cell surface. After this procedure, the percentage positivity of collagen type IV detected by flow cytometry was markedly reduced to <3%. Dishes were coated with human collagen type IV as described in the manual instructions. Briefly, human collagen type IV (Becton Dickinson Labware, Bedford, MA, USA) was dissolved at a concentration of 10 µg/ml in 10 mM acetic acid, and 200 µl was placed in each well of 96-well non-coated polystyrene dishes (Corning Glass Works, Corning, NY, USA). The dishes were incubated at room temperature for 2 h, and washed in PBS before use. The granulosa cells isolated as described above were suspended in Dulbecco's modified Eagles medium (DMEM)/Ham's F-12 (1:1 v/v, Gibco BRL) medium containing 5% fetal calf serum (FCS; Flow Laboratories, McLean, VA, USA) and 10 mM HEPES (Nacalai Tesque, Kyoto, Japan) without antibiotics, at a density of 3x105 cells/ml. The cells were inoculated into 96-well non-coated, or human collagen type IV-coated polystyrene dishes at 100 µl/well. After 24 h (day 1), the media were exchanged with non-FCS containing media. The cells were cultured for 3 days in the absence or presence of HCG (1 IU/ml, Serono Japan, Tokyo, Japan). The media were changed every 24 h and were collected to assay steroid hormone production. To assay oestradiol, 10–7 M testosterone was added to the medium. In the non-coated group, the concentration of collagen type IV in the media was also measured. The cells were detached by incubation with PBS containing 0.05% trypsin (Difco, Detroit, MI, USA) and 0.05% EDTA (Nacalai Tesque), then the number of viable granulosa cells per well was counted under microscopy by Trypan Blue exclusion.
In some cases, after collagenase treatment granulosa cells were seeded into 8-well glass slide chambers (LabTek Nunc Inc, Naperville, IL, USA) for immunocytochemistry, and cultured for 4 days as described above. On day 1 or day 4, the medium was discarded and the attached cells on the slides were air-dried and fixed by acetone at –20°C for 5 min. The slides were stained with anti-integrin 2 and anti-collagen type IV mAb as described above.
RNA isolation
The granulosa cells freshly isolated from IVF-treated patients were immediately frozen in liquid nitrogen and stored at –80°C until RNA extraction. Total RNA of these tissues were isolated using a commercial kit (TRIzol; Gibco BRL, Gaithersburg, MD, USA).
Reverse transcription–polymerase chain reaction (RT–PCR) analysis of collagen type IV mRNA in the granulosa cells
Total RNA (5 µg) from luteinizing granulosa cells was reverse-transcribed with random primers by a commercial kit (First Strand cDNA Synthesis Kit; Pharmacia Inc, Piscataway, NJ, USA). The resulting cDNA mixtures were subjected to 30 cycles of PCR amplification with oligonucleotides from the human collagen type IV cDNAs as primers (sense primer 5'-GTGGGTTTTCTTTTCTTTTT-3': position 4567–4587; antisense primer 5'-CACCTGACAGCGACTTATTA-3': position 5134–5154) for isotype 3 (Mariyama et al., 1994), (sense primer 5'-GGTGGCTTCCTCCTGGTTCT-3': position 4598–4618; antisense primer 5'-GGCTGATTTTCTGGCGTTGG-3': position 5233–5253) for isotype 4 (Leinonen et al., 1994), and (sense primer 5'-ATCCTGGTCTCCCTGGTGTT-3': position 1226–1246; antisense primer 5'-GCATCCGTTGTCTGGCTGTG-3': position 1660–1680) for isotype 5 (Zhou et al., 1991), or with human S26 primers (sense primer 5'-GGTCCGTGCCTCCAAGATGA-3': position 8–27; antisense primer 5'-TAAATCGGGGTGGGGGTGTT-3': position 308–327) (Vincent et al., 1993). After PCR amplification, 10 µl from each PCR product was electrophoresed on a 1% agarose gel, and amplified bands were detected by ethidium bromide staining.
Retrieval of oocytes and follicular fluid
Oocytes and follicular fluid were retrieved from the patients (n = 29), aged 24–41 years, who underwent intracytoplasmic sperm injection (ICSI). Ovulation induction was performed as described above. The samples of follicular fluid were obtained from one or two follicle(s) per patient and were immediately centrifuged at 1000 g for 10 min, and the supernatant was stored at –80°C until assay.
Oocyte morphology
The cumulus–oocyte complex was treated with 0.05% hyaluronidase 2 h after oocyte retrieval and the cumulus-removed oocyte was observed under light microscopy (Nikon, Tokyo, Japan) at the time of ICSI. Only mature oocytes (metaphase II) were included in this study, and immature oocytes or empty follicles were excluded. Oocyte cytoplasm morphology was classified as previously (Veeck, 1988, 1991). Cytoplasm with excessive granularity, with large perivitelline spaces, with central darkness, or with cytoplasmic vacuole were classified as abnormal. Cytoplasm without these findings were classified as normal.
All the oocytes were inseminated by ICSI and were incubated in -modified Eagle's medium (ICN Biomedicals Inc, Ohio, USA) with 1% human albumin (Green Cross Corporation, Osaka, Japan) in 5% CO2, 5% O2 at 37°C.
Grading and scoring of embryo morphology
The embryos were observed on day 2, shortly before embryo transfer. Morphology of embryos were graded from G1 to G5 using Veeck's classification (Veeck, 1988, 1991), and were scored as from 1 (G1) to 5 (G5) using inverted microscopy. Embryos with delayed cleavage, i.e. fewer than 4 cells at day 2, were scored as 5.
Assay of steroid hormone and collagen type IV production by human luteinizing granulosa cells
The concentrations of progesterone and oestradiol were measured using radioimmunoassay kits (Daiichi Radio Isotope Research Inc, Tokyo, Japan). Inter- and intra-assay coefficients of variation were 6.5 and 5.3% for the progesterone assay and 7.4 and 6.3% for the oestradiol assay respectively. The concentration of collagen type IV was measured by an enzyme-linked immunosorbent assay (ELISA) kit (Daiichi Pure Chemicals Co Ltd, Tokyo, Japan).
Statistical analysis
Each experiment was performed in triplicate. Data are shown as mean ± SEM. Simple regression analysis was used for the relationship between steroid hormone and collagen type IV concentrations and the relationship between embryo scores and collagen type IV concentrations. Production of collagen type IV in the granulosa cell culture was analysed by the two-tailed paired t-test. Differences of collagen type IV concentrations in the follicular fluid between normal and abnormal groups of oocyte morphology were analysed by the two-tailed unpaired t-test.
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Expression profiles of integrin
2 and collagen type IV in the human ovaryIn growing follicles (3–13 mm in diameter, n = 6), integrin
2 was highly expressed on granulosa cells in all layers, whereas it was not detected on theca interna and stromal cells (Figure 1). On the other hand, collagen type IV was highly expressed around theca interna and endothelial cells, but not around granulosa cells.
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In pre-ovulatory follicles (18–20 mm in diameter, n = 4), integrin
2 was highly expressed on the granulosa cells, and collagen type IV was also detected patchily, but clearly, among the granulosa cells including those located in the inner layers (Figure 2). In theca interna layer, collagen type IV was also clearly detected among theca interna cells, whereas integrin 2 was not detected on these cells. These expression profiles of integrin 2 and collagen type IV in the follicles are summarized in Table I.
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In CL of the early luteal phase (CL day 2: n = 2, day 3: n = 1, day 4: n = 2, day 5: n = 2), integrin
2 continued to be highly expressed on luteinizing granulosa cells (Figure 3). The expression intensity of collagen type IV among luteinizing granulosa cells was rapidly increased after ovulation. Additionally, high expression of integrin 2 was also observed on luteinizing theca interna cells, being concomitant with high expression of collagen type IV among these cells.
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In CL of the mid-luteal phase (CL day 7: n = 3, day 8: n = 3, day 9: n = 2), integrin
2 was highly expressed on large luteal cells and highly or weakly on small luteal cells (Figure 4). Collagen type IV was highly detected around both large and small luteal cells.
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In CL of the late luteal phase (CL day 13: n = 3, day 14: n = 3), the expression of integrin
2 was decreased to be none or weak on both large and small luteal cells. Conversely, collagen type IV was clearly expressed around both luteal cells (data not shown).
In CL of early pregnancy (6, 7, 9, 11 and 14 weeks of gestation: n = 5), integrin 2 was hardly detected on large and small luteal cells, whereas collagen type IV was clearly observed around both luteal cells (Figure 5). These expression profiles of integrin 2 and collagen type IV in the CL are summarized in Table II.
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Flow cytometric analysis of isolated granulosa cells
Integrin
2 and collagen type IV were detected on the cell surface of granulosa cells isolated from the patients undergoing IVF treatment. Flow cytometry showed that integrin 2, integrin ß1, and collagen type IV were expressed on analysed granulosa cells at the rates of 91.5 ± 6.8%, 84.7 ± 9.6%, and 41.2 ± 10.8% respectively (n = 5, mean ± SD) (Figure 6).
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The expression of integrin
2 and the production of collagen type IV in cultured granulosa cellsImmunocytochemical staining showed no apparent alteration of the expression of integrin
2 on granulosa cells during 4 day culture, whereas the expression of collagen type IV around them were obviously augmented during the culture (Figure 7).
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RT–PCR analysis of collagen type IV mRNA in the granulosa cells
The expressions of collagen type IV mRNA (isotype 3, 4 and 5) were observed in the granulosa cells and CL (Figure 8
). The nucleotide sequence of the PCR products in granulosa cells were analysed by DNA sequencing and confirmed to be identical to those of collagen type IV cDNAs as previously reported (Zhou et al., 1991; Leinonen et al., 1994; Mariyama et al., 1994). The expected PCR products of S26 were also detected in the granulosa cells and CL.
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Effect of HCG on the production of collagen type IV by cultured granulosa cells
The concentrations of collagen type IV in the culture media were significantly higher in the HCG-treated group than that in the non-treated group (Figure 9
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Effects of collagen type IV on progesterone and oestradiol production by human luteinizing granulosa cells
After 4-day culture, there was no difference in cell morphology and the number of viable human luteinizing granulosa cells between the groups cultured on collagen type IV-coated and non-coated dishes. In the absence or presence of HCG, progesterone production cultured on collagen type IV-coated dishes was 0.69- and 0.75-fold, compared with those on non-coated dishes (P < 0.05 respectively, Figure 10A
). There was no significant difference in oestradiol production between the two groups (Figure 10B).
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Relation between collagen type IV and steroid hormone concentrations in the follicular fluid
There was a significant correlation between collagen type IV and progesterone concentrations in the follicular fluid (P < 0.01, Figure 11
), whereas no correlation was observed between collagen type IV and oestradiol (data not shown).
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Relation between collagen type IV concentration in the follicular fluid and oocyte or embryo morphology
There was no difference in collagen type IV concentration between the normal ooplasm group and the abnormal group (Figure 12
). Similarly, there was no correlation between morphological embryo grade and the follicular fluid concentration of collagen type IV (data not shown).
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Discussion |
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Integrin
2 belongs to an integrin supergene family of cell adhesion receptors and constitutes a heterodimer with integrin ß1, which is identical to the platelet membrane glycoprotein Ia–IIa complex (Pischel et al., 1988; Hynes, 1992). Integrin 2ß1 is a major collagen receptor in fibroblasts and platelets (Wayner and Carter, 1987; Takeda et al., 1988) and is involved in platelet aggregation induced by collagen (Nieuwenhuis et al., 1985). Integrin 2ß1 is also involved in the migration of tumour cells via the interaction with collagen (Yamada et al., 1990). It was to date reported that ligands for integrin 2ß1 include collagen type I–IV and VI, laminin, and echovirus 1 (Wayner and Carter, 1987; Elices and Hemler, 1989; Bergelson et al., 1992; Shaller and Parsons, 1994).
In this study, we showed by immunohistochemistry that integrin 2 was expressed on granulosa cells in the growing and pre-ovulatory follicles. The high expression of integrin 2 on granulosa cells continued during the luteinization process after ovulation until they differentiate into large luteal cells, and then integrin 2 expression was decreased or disappeared in the late luteal phase. When menstrual CL differentiated into CL of pregnancy, the expression of integrin 2 on large luteal cells was attenuated. Immunostaining of isolated granulosa cells demonstrated the presence of integrin 2 on the cell surface, and flow cytometry revealed that >90% of the analysed cells express integrin 2. From these findings, we concluded that integrin 2 is a differentiation-related cell surface molecule of human granulosa cells. Previously, we reported that integrin 6ß1 is also a differentiation-related cell surface molecule of human granulosa cells (Honda et al., 1995). Additionally, we found that integrin 5 rapidly appears on the cell surface of human granulosa cells during ovulation and the expression is augmented by HCG administration, indicating integrin 5 to be an initial luteinizing marker of granulosa cells (Honda et al., 1997). Thus, granulosa cells were shown to bear several integrins on the cell surface in accordance with their differentiation stages, suggesting that the switching of integrin expression is involved in granulosa cell differentiation. On the other hand, integrin 2 began to be expressed on luteinizing theca interna cells after ovulation. This indicates that integrin 2 is also a differentiation-related molecule for theca interna cells and suggests the implication of integrin 2 in the process of differentiation of theca interna cells into small luteal cells.
Previously, integrin 6ß1 was demonstrated to suppress luteinization of human granulosa cells via the interaction with laminin, suggesting that integrin 6ß1 is a new regulator of luteinization of human granulosa cells during corpus luteum formation (Fujiwara et al., 1997). On the other hand, HCG and extracellular matrices were demonstrated to synergistically regulate human granulosa cell differentiation, proposing an important role for extracellular matrices in ovarian physiology (Amsterdam et al., 1989). Laminin was rapidly increased among luteinizing human granulosa cells after ovulation (Fujiwara et al., 1997). In addition, the expression of integrin 5 on luteinizing human granulosa cells after ovulation is also accompanied by the rapid appearance of its ligand fibronectin (Honda et al., 1997). From these findings, it is postulated that the extracellular matrices play a physiological role in granulosa cell function in concert with integrins. Therefore, we examined immunohistological distribution of collagen type IV, which is one of ligands for integrin 2ß1 (Shaller and Parsons, 1994), in the human ovary.
While collagen type IV was not detected in the granulosa cell layers of the growing follicles by immunohistochemistry, it was highly expressed among luteinizing granulosa cells in the post-ovulatory follicles. Collagen type IV was shown to bind to the cell surface of the granulosa cells isolated from patients undergoing IVF by immunostaining and flow cytometry, indicating the physiological interaction between collagen type IV and granulosa cells prior to ovulation. This interaction may be partially due to integrin 2ß1. The concentration of collagen type IV in the pre-ovulatory follicles was shown to be significantly and positively correlated with follicular progesterone concentration. After the granulosa cells were denuded of collagen type IV by collagenase treatment, collagen type IV began to be detected again in the media and among granulosa cells during the 4-day culture in vitro. In addition, mRNAs of isotypes 3, 4, and 5 of collagen type IV were shown to be expressed in luteinizing granulosa cells. Furthermore, the concentration of collagen type IV in the media was enhanced by HCG treatment, showing that the production of collagen type IV is regulated by luteinizing hormone (LH)/HCG. These findings are compatible with immunohistochemical profiles of collagen type IV expression in the pre- and postovulatory follicles. Thus, we considered that granulosa cells produce collagen type IV under the stimulation of LH and the secreted collagen type IV interacts with luteinizing granulosa cells in an autocrine fashion during ovulation and CL formation. It can be also proposed that the production of collagen type IV is a new parameter of granulosa cell luteinization.
To elucidate the physiological role of collagen type IV in ovarian cell function, we examined the effect of collagen type IV on progesterone production by luteinizing granulosa cells and investigated the correlation between oocyte morphology and collagen type IV concentration in the follicular fluid. In the collagen type IV-coated dishes, progesterone production by luteinizing granulosa cells was attenuated as compared with those cultured in the non-coated dishes. This indicates that collagen type IV suppresses granulosa cell luteinization to serve as a local regulator for luteinization (Fujiwara et al., 1997). During the LH surge, granulosa cell differentiation is deeply related with the processes of oocyte meiosis and follicular rupture. The alteration in the granulosa cell function induced by LH surge is considered to play an essential role in the resumption of oocyte meiosis (Wassarman, 1996). The augmented production of progesterone in granulosa cells induced by the LH surge is also inevitable as a trigger for the inflammatory cascade of follicular rupture (Mori et al., 1977; Iwamasa et al., 1992). These two processes are co-ordinated to prepare a metaphase II stage in the oocyte when it is expelled into the pelvic cavity. Additionally, the maturation of cumulus granulosa cells during ovulation, which surround the oocyte and are expanded with proteoglycans, is important for the retrieval of the oocyte–cumulus complex by the Fallopian fimbria (Salustri et al., 1992; Camaioni et al., 1996). Thus, for successful fertilization, the pace of granulosa cell differentiation during ovulation should be strictly regulated to orchestrate oocyte meiosis and follicular rupture. At present, LH/HCG is the only established promoting factor for granulosa cell differentiation in the periovulatory phase. Collagen type IV produced by luteinizing granulosa cells may be one of the candidates for this regulation. In addition to collagen type IV, laminin was also demonstrated to be increased among granulosa cells during ovulation (Fujiwara et al., 1997) and its follicular concentration was positively correlated with progesterone concentration (unpublished results). Since both collagen type IV and laminin suppressed progesterone production by luteinizing granulosa cells (Fujiwara et al., 1997), we speculate that their concentrations in the follicular fluid are increased during luteinization of granulosa cells in pre-ovulatory follicles, and then the differentiation of granulosa cells is negatively regulated in an autocrine fashion by these extracellular matrices to protect the accelerated luteinization.
Recently, we found that fibronectin, but not laminin, in the follicular fluid was significantly higher in the normal oocyte morphology group than in the abnormal group, indicating that fibronectin is a new parameter for oocyte quality (unpublished results). This study showed that the collagen type IV concentration was not significantly related with oocyte nor embryo morphology. These observations suggest the differential roles of extracellular matrices in oocyte and granulosa cell function.
In contrast to laminin and fibronectin (Fujiwara et al., 1997; Honda et al., 1997), collagen type IV was patchily, but clearly, detected among granulosa cells in the pre-ovulatory follicles by immunohistochemistry. This suggests that collagen type IV functions as a regulator for granulosa cell luteinization at an earlier stage than laminin. Before the initiation of the LH surge, the prevention of premature luteinization is another important issue which influences periovulatory events. Even after acquisition of LH receptor, the maturing granulosa cells in the follicles should not easily respond to normal concentrations of serum LH until the real central signal, i.e. the LH surge, is started. Although it is still controversial, premature luteinization has been reported to be associated with a poor pregnancy outcome (Schoolcraft et al., 1991; Silverberg et al., 1991). Collagen type IV produced among granulosa cells may be involved in the inhibition of premature luteinization before the LH surge.
In conclusion, this study showed that integrin 2 is a differentiation-related molecule of human granulosa and theca interna cells, and that its ligand, collagen type IV, is rapidly produced around luteinizing granulosa cells during ovulation and interacts with the cell surface of granulosa cells. Collagen type IV was produced by granulosa cells and its production was augmented by HCG in vitro. The concentration of collagen type IV in the follicular fluid was positively correlated with that of progesterone, indicating that collagen type IV is a new parameter of granulosa cell luteinization. Furthermore, collagen type IV suppressed progesterone production by luteinizing granulosa cells in vitro. These findings suggest that granulosa cells produce collagen type IV under the stimulation of LH and the secreted collagen type IV interacts with luteinizing granulosa cells partially via integrin 2ß1 in an autocrine fashion to regulate the differentiation of granulosa cells during ovulation and CL formation.
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Acknowledgments |
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The authors are grateful to Mrs Hisako Takahashi, Mrs Nami Kohama and Miss Chikako Kataoka, for technical assistance in IVF/ICSI and RIA. This work was supported in part by Grants-in-Aid for Scientific Research, (no. 09671673, 09671674, 09671676).
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4 To whom correspondence should be addressed
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References |
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Submitted on December 8, 1998; accepted on March 31, 1999.
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