Department of Obstetrics and Gynecology, Kansai Medical University, 1015 Fumizono-cho, Moriguchi, Osaka, 570-8507, Japan
1 To whom correspondence should be addressed. Email: nakamott{at}takii.kmu.ac.jp
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Abstract |
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Key words: endometrial stromal cell culture/fibulin-1/human endometrium/progesterone
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Introduction |
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To investigate the molecular mechanisms underlying decidualization during the preparatory period of implantation, we attempted to identify the genes induced by progesterone in ESCs in short-term culture (3 days, using microarray analysis), and demonstrated that one of the genes up-regulated after progesterone treatment was fibulin-1 (Okada et al., 2003), which codes for an extracellular matrix (ECM) and plasma glycoprotein, and has been implicated in tumour progression (Qing et al., 1997
; Hayashido et al., 1998
; Twal et al., 2001
). Recently, it is suggested that fibulin-1 in ESCs and endometrium is induced by progesterone (Haendler et al., 2004
), but the roles of fibulin-1 in human endometrium, particularly during differentiation process towards implantation, have not been well studied. In the present investigation, we examined the localization and spatial and temporal hormonal regulation of fibulin-1 in human endometrial tissues. In addition, using an in-vitro culture system of human ESCs, we precisely investigated fibulin-1 gene expression during decidualization induced by 6
-methyl-17
-hydroxy-progesterone acetate (MPA) as well as 8 bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP).
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Materials and methods |
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Immunohistochemistry
Eleven secretory phase and four proliferative phase endometrial tissue samples were fixed for 18 h at room temperature with 10% formalin neutral buffer solution. After fixation the tissues were placed in 70% ethanol overnight at 4 °C, then embedded in paraffin and sectioned at 4-mm thickness. Tissue sections were then mounted on 3-aminopropyltriethoxy-silane-coated glass slides. Next, the tissue sections were deparaffinized in xylene then rehydrated in a graded alcohol series. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol. The sections were then washed in phosphate-buffered saline (pH 7.2), and microwave antigen retrieval procedure using 10 mmol/l citrate buffer solution (pH 6.0) (Muto Pure Chemical Co. Ltd, Tokyo, Japan) was performed for 10 min. After microwave exposure, the slides were allowed to cool to room temperature. The slides were incubated for 15 min in 10% normal horse serum prior to the application of the primary antibody. The streptavidinbiotin complex peroxidase (SAB) method (Histofine SAB-PO kit; NICHIREI Corp., Tokyo, Japan) was used. Incubation with a rabbit polyclonal antibody against fibulin-1 (H-190) (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was performed for 60 min at room temperature. This antibody recognises all four fibulin-1 forms. Antibody staining was developed with diaminobenzidine for 5 min. The sections were then counter-stained with haematoxylin, dehydrated in a graded alcohol series, cleared in xylene, and finally mounted in balsam. Negative control sections were treated identically, except that the specific antibody was replaced by the same concentration of normal rabbit serum.
Fibulin-1 expression was evaluated under a double-headed microscope at 200 x magnification. The number of positive cells was counted in each of endometrial stromal and glandular epithelial portions. Staining density was quantified as the percentage of cells staining positive with the primary antibody, as follows: 0 = no staining, 1 = superficial staining, 2 = positive staining in <25% of the sample, 3 = positive staining in 2550% of the sample, 4 = positive staining in >50% of the sample and 5 = positive staining throughout the sample (Roark et al., 1995; Amara et al., 2001
). The menstrual phase of the specimens was classified based on the analysis of haematoxylineosin-stained sections using conventional histological criteria (Noyes et al., 1950
).
RNA isolation and deoxyribonuclease I treatment
Total RNA was prepared from endometrial tissues or cultured ESCs by the acid guanidinium phenol-chloroform method using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and dissolved in appropriate amount of 0.01% diethyl pyrocarbonate water. Spectrophotometric determination of the quality and quantity of RNA was assessed at OD 260 nm and 280 nm (the absorbance ratio was >1.8).
Two micrograms of each RNA sample were incubated for 15 min at room temperature in a volume of 20 ml, containing 2 ml of 10xdeoxyribonuclease I (DNase I) buffer [200 mmol/l TrisHCl (pH8.4), 20 mmol/l MgCl2, 500 mmol/l KCl], 2 ml of DNase I Amplification Grade (Invitrogen) and 1 ml of RNase Inhibitor (Wako Pure Chemical Co. Ltd). Following the incubation, the reaction was terminated by the addition of 2 ml of 25 mmol/l EDTA solution, and heated for 10 min at 65 °C to inactivate the enzyme.
Reverse transcription
Reverse transcription (RT) was performed using the ReverTra Ace Kit (TOYOBO Co., Ltd, Tokyo, Japan) according to the manufacturer's instructions: 1 mg of total RNA treated with DNase I in 20 ml of reaction mixture [final concentrations: 50 mmol/l TrisHCl (pH 8.3), 75 mmol/l KCl, 3 mmol/l MgCl2, 10 mmol/l dithiothreitol (DTT), 0.1 mmol/l random primer, 1 mmol/l deoxytimidine triphosphate (dTTP), 0.3 mmol/l (methyl-3H) dTTP, 1 mmol/l deoxynucleotide mixture (dNTP)] containing 100 U of ReverTra Ace Reverse Transcriptase at 30 °C for 10 min, at 42 °C for 20 min, followed by inactivation of the enzyme at 99 °C for 5 min with a TaKaRa PCR Thermal Cycler MP (TAKARA SHUZO, Kyoto, Japan). Finally, the first-strand cDNA was dissolved in 80 ml of distilled water and stored at 20 °C. The control reaction was performed simultaneously under identical conditions, but without reverse transcriptase.
Real-time PCR analysis with SYBR Green I
For quantitation of fibulin-1 and prolactin (PRL) mRNA, real-time quantitative PCR using the SYBR Green I nucleic acid gel stain (Roche Diagnostics GmbH, Mannheim, Germany) was performed in triplicate on 96-well optical reaction plates (INA. OPTIKA Co., Ltd, Osaka, Japan) using an iCycler iQ (Bio-Rad, Tokyo, Japan). The PCR was performed in a total volume of 25 ml containing 2 ml of the above-described cDNA, 1 ml each of the 3' and 5' primers (3.75 mmol/l each), 1 ml MgCl2 (25 mmol/l), 2 ml dNTP (2.5 mmol/l), 2.5 ml 10 x GeneTaq Universal Buffer (Wako Pure Chemical Co. Ltd), 0.375 U recombinant Taq DNA polymerase GeneTaq (Wako Pure Chemical Co. Ltd), 0.075 ml monoclonal antibody for Hot Start PCR anti-Taq high (TOYOBO Co., Ltd) and 1/75 000 SYBR Green I nucleic acid gel stain. PCR amplification of each sample was performed on the same reaction plate using both the human fibulin-1 and PRL gene primer pairs, together with primers for the elongation factor-1 (EF-1
) gene, which served as an internal control. EF-1
is valid as a reference housekeeping gene for transcription profiling, which is also used for real-time PCR experiments (Frost and Nilsen, 2003
). The oligonucleotide primers were synthesized by Proligo Japan (Kyoto, Japan). The fibulin-1 oligonucleotide primer sequences were 5'-TGCTTCGTGGGCTACCAGCTGCTGT-3' (forward) and 5'-CTCCTCGTTGAGATGGTAGCCACGG-3' (reverse) (Argraves et al., 1990
; Tran et al., 1997a
). These primers amplified a 450-bp fragment of human fibulin-1 cDNA, which is the common sequence among the four variants (A to D) of fibulin-1 (bases 6111060 of fibulin-1D) (Argraves et al., 1990
; Tran et al., 1997a
; Pan et al., 1999
). The PRL oligonucleotide primer sequences were 5'-ATTCGATAAACGGTATACCCATGGC-3' (forward) and 5'-TTGCTCCTCAATCTCTACAGCTTTG-3' (reverse) (Strausberg et al., 2002
). These primers amplified a 250-bp fragment of human PRL cDNA (bases 723972). The EF-1
oligonucleotide primer sequences were 5'-TCTGGTTGGAATGGTGACAACATGC-3' (forward) and 5'-AGAGCTTCACTCAAAGCTTCATGG-3' (reverse) (Strausberg et al., 2002
; Doi et al., 2002
). These primers amplified a 329-bp fragment of EF-1
cDNA (bases 595923). The PCR comprised 50 cycles: 94 °C for 30 s, 72 °C for 60 s (set-point temperature was decreased every two cycles by 0.3 °C), and 72 °C for 30 s. After PCR, a melting curve was constructed by increasing the temperature from 65 °C to 95 °C with a temperature transition rate of 0.5 °C/30 s. After melting curve analysis, the concentration of each sample was calculated from the threshold cycle (Ct). To facilitate the comparison of fibulin-1 and PRL mRNA expressions, the fibulin-1 and PRL values from each sample were normalized by the EF-1
Ct value obtained from that same sample. The Ct values were averaged for each triplicate. Differences between the mean Ct values of fibulin-1 (or PRL) and those of EF-1
were calculated, as follows:
Ct (sample) = Ct (sample)Ct (EF-1
). For real-time PCR experiments for mRNAs from seven secretory phase endometrial tissues (from day 19 to day 28 of the menstrual cycle) and seven proliferative phase endometrial tissues (from day 6 to day 15 of the menstrual cycle), final result was expressed as 2
Ct sample. The N-fold increase or decrease in expression for cultured ESCs experiments was calculated by
Ct method with control Ct value as the reference point, which was obtained from the ESCs before any drug addition. N-fold difference was determined as 2(
Ct sample
Ct control) (Somasundaram and Bhat, 2004
). In order to eliminate the possibility of contamination with genomic DNA during extraction of total RNA, a control reaction with each primer pair was performed at the same time under identical conditions without reverse transcription, and no amplification was detected.
Isolation and culture of endometrial stromal cells
Endometrial stromal cells (ESCs) were isolated as described previously (Okada et al., 1999; Okada et al., 2000
). Briefly, tissue samples were washed with Dulbecco's modified Eagle's medium (DMEM)/F-12 medium (Invitrogen) and minced into small pieces of <1 mm3. The tissue was then incubated for 2 h at 37 °C in DMEM/F-12 medium containing 1 mg/ml collagenase (Wako Pure Chemical Co. Ltd) and 0.005% DNase I (Boehringer Mannheim GmbH, Mannheim, Germany). After subsequent pipetting, the cell suspension was diluted with two volumes of DMEM/F-12 medium and placed in a centrifugation tube (Corning, Inc., Corning, NY, USA), which remained upright for 10 min at unit gravity. The supernatant, excluding the lowermost 2 ml, was transferred into a new tube to collect suspended single cells. After repeating this procedure several times, the cell suspension was washed three times and used as a source of ESCs. The viability, determined by Trypan Blue dye exclusion, was at least 90%. Two million viable ESCs were cultured in 75 cm2 flasks in DMEM/F-12 medium supplemented with 10% fetal calf serum (FCS) (HyClone Laboratories, Inc., Logan, UT, USA), 100 IU/ml penicillin and 100 mg/ml streptomycin (Invitrogen) at 37 °C in humidified atmosphere of 5% CO2 in air. The culture medium was replaced 30 min after plating to reduce epithelial cell contamination. The purity of ESCs was determined by morphology and by immunohistochemical staining, as described previously (Inoue et al., 1996
), with markers specific to ESCs (vimentin), epithelial cells (cytokeratin), endothelial cells (factor VIII) or leukocytes (CD45). These antibodies were purchased from DAKO Corp. (Kyoto, Japan) and cells were transferred to Lab-Tek chamber slides (Nalge Nunc, Naperville, IL, USA) for immunohistochemical staining. Initially, the purified fraction contained approximately 12% endothelial cells, 23% epithelial cells, 12% leukocytes, 12% macrophages and 95% ESCs by immunohistochemistry. The proportion of vimentin-positive cells in confluent ESCs was >99% by immunohistochemical staining.
In-vitro decidualization of ESCs and steroid hormone treatment
After 12 weeks culture, when ESCs were nearly confluent, cells were plated in 75 cm2 flasks for western blot analyses and in six-well plates for real-time PCR analyses. To remove the effect of endogenous steroid hormones, the FCS to be used in the cell cultures was treated as follows. FCS (100 ml) mixed with 0.25 g activated charcoal (SigmaAldrich Co., St Louis, MO, USA) and 0.025 g dextran (clinical grade; SigmaAldrich Co.) was stirred at 56 °C for 30 min and centrifuged to separate the dextran-coated charcoal pellet. The supernatant was then subjected to the some treatment at 37 °C and the dextran-coated charcoal-stripped (DCS)-FCS was filtered through a 0.45 mm sterilization unit (Corning Inc.) and stored at 20 °C. ESCs were cultured until confluent, when the media were replaced with phenol red-free DMEM/F-12 supplemented with 10% DCS-FCS. After 48 h, ESCs were washed and then cultured in DCS-FCS media supplemented with MPA (SigmaAldrich Co.), -estradiol (E2) (Wako Pure Chemical Co. Ltd), RU-486 (SigmaAldrich Co.), 8-Br-cAMP (SigmaAldrich Co.) or dimethyl sulfoxide (Wako Pure Chemical Co. Ltd) as a vehicle control. Additive-free ESCs cultured in DCS-FCS media were used as a control for real-time PCR. The culture media were changed every 3 days.
Western blot analysis
For analysis of fibulin-1 protein levels, cultured cells that were incubated under differing hormonal conditions were homogenized in lysis buffer containing 50 mmol/l TrisHCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 50 mmol/l DTT and 0.1% Bromophenol Blue, and diluted to 1 mg total protein/ml. Samples (10 mg) were resolved by 10% SDSPAGE. Proteins were blotted onto sequi-blotTMPVDF Membrane (Bio-Rad) and incubated with anti-fibulin-1 (H-190) (1:500; Santa Cruz Biotechnology, Inc.) or monoclonal anti--actin (SigmaAldrich Co.) as a primary antibody, and anti-rabbit IgG peroxidase-labelled secondary antibody (1:5000; KPL Inc., Gaithersburg, MD, USA) or anti-mouse IgG peroxidase-labelled secondary antibody (1:10 000; KPL Inc.) as a secondary antibody. Immune complexes were visualized by use of a LumiGLO Chemiluminescent Substrate (Cell Signaling Technology, Beverly, MA, USA).
Statistical analysis
Data are expressed as mean ± standard error of the mean (SEM). Real-time PCR experiments for human endometrium and immunohistochemical score were statistically assessed using conservative non-parametric statistics, the MannWhitney test. Real-time PCR experiments for ESCs sample were repeated in a minimum of six independent experiments, and for those experiments differences in the measured parameters across the different groups were statistically assessed using ANOVA with repeated measurements, followed by Fisher's protected least significant difference, multiple range test. Results were analysed with the statistical software package StatView, version 5.0 (SAS Institute Inc., Cary, NC, USA). A P-value < 0.05 was considered statistically significant.
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Results |
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Discussion |
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By microarray analysis, we have isolated and demonstrated that fibulin-1 mRNA is induced by progesterone in human ESCs in short-term culture, prior to the morphological cellular transformation (Okada et al., 2003). Two other reports have shown that fibulin-1 expression is regulated by hormonal control in endometria of several species (Allan et al., 2003
; Haendler et al., 2004
). However, in those reports the hormonal regulation of fibulin-1 mRNA expression in ESCs and the localization of fibulin-1 protein in the human endometrium during menstruation and implantation process were not described clearly. Furthermore, the spatial and temporal regulation of fibulin-1 by progesterone in human endometrium has been more controversial.
The present study showed that fibulin-1 mRNA levels in human endometrial tissues were increased significantly during the secretory phase rather than the proliferative phase, which was suggested by a previous report with a small sample size, using qualitative northern blot analysis (Haendler et al., 2004). To analyse the data statistically, we performed real-time PCR experiences on fibulin-1 mRNA and found that the fibulin-1 mRNA expression level is augmented during the menstrual cycle in human endometrium as a whole, which includes the glandular epithelium and stromal components. Immunohistochemical analysis revealed that fibulin-1 protein expression switched from the glandular epithelial cells during the proliferative phase to the stromal cells during the secretory phase, which is consistent with the report by Allan et al. (2003)
, who carried out an in-situ hybridization analysis of cynomolgus monkey.
In addition, using an in-vitro culture system of human ESCs, we confirmed that the fibulin-1 mRNA expression is apparently induced by progesterone in ESCs after 3 days of culture, which previously was suggested by microarray method (Okada et al., 2003). After 3 days of culture, fibulin-1 mRNA expression induced by progesterone continued to increase until the end of the 15-day culture period. Fibulin-1 mRNA expression by progesterone was dose-dependent and the progesterone effect was completely attenuated by the anti-progestin RU-486. On the other hand, E2 alone had no effect on fibulin-1 mRNA expression. In a previous report, the expression levels of fibulin-1 using the ESCs with several conditions by western blotting analysis suggested qualitatively that progesterone induces the fibulin-1 expression, but that E2 does not influence fibulin-1 expression (Haendler et al., 2004
). The present findings confirmed quantitatively these hormonal influences toward the fibulin-1 expressions using a different method, real-time PCR analysis. Interestingly however, fibulin-1 expression in the condition medium was spontaneously raised during the cultures in the absence of progesterone, which may suggest the existence of another regulator for fibulin-1. In-vitro decidualization can be induced by two pathways, one progesterone-mediated and the other cAMP-mediated (Mizuno et al., 1998
; Hwang et al., 2002
). In the light of another cAMP-mediated pathway, we also investigated 8-Br-cAMP and found that it induced fibulin-1 mRNA expression in ESCs after 3 days of culture. In the present study, fibulin-1 protein in ESCs following treatment with progesterone was more abundant than that following treatment with E2 or vehicle after 15 days of culture by qualitative western blot analysis, which is consistent with mRNA levels by quantitative real-time PCR analyses. These in-vitro and in-vivo findings confirmed that the ovarian steroid hormone progesterone strongly induced fibulin-1 expression in the stroma of the human endometrium during decidualization.
Several studies have reported that estrogen increases the expression of fibulin-1 in several ovarian cancer cell lines (Clinton et al., 1996; Moll et al., 2002
), and that fibulin-1 expression is up-regulated in the rat endometrium after hormonal treatment with E2 (Haendler et al., 2004
). ESCs and the glandular epithelial cells of human endometrial tissues are known to have estrogen receptors (Brandenberger et al., 1999
), but E2 did not affect fibulin-1 expression in cultured ESCs. On the other hand, fibulin-1 protein in the glandular epithelial cells of endometrium was more abundantly expressed during the E2-dominant proliferative phase than in the progesterone-dominant secretory phase. Thus, the exact biological function of fibulin-1 in the uterus is difficult to describe because of the presence of both epithelial and stromal cells, which exhibit different expression patterns of the molecule probably due to the different responses to estrogens and progesterone.
Fibulin-1 has been shown to self-associate as well as to bind calcium and other ECM proteins including fibronectin, laminin, nidogen and fibrinogen (Tran et al., 1997b). The ECM and integrins, which participate in cellcell adhesion as well as in adhesion between cells and components of the ECM, play an important role(s) in embryo implantation (Bischof and Campana, 2000
; Merviel et al., 2001
). The expression levels of several components of the ECM, collagen VI (Boos, 2000
), laminin (Faber et al., 1986
) and fibronectin (Zhu et al., 1992
), are known to be regulated in the uterus. Integrins are adhesion molecules that participate in cellcell adhesion as well as in adhesion between cells and components of ECM. Indeed, expression of several integrins in the cytotrophoblast have been described (Bischof and Campana, 2000
; Merviel et al., 2001
), and Damsky et al. (1992)
have described that the villous cytotrophoblast and the invasive extravillous cytotrophoblast express different integrins. Furthermore, several reports have demonstrated that fibulin-1, together with fibronectin, suppresses cell adhesion and motility (Hayashido et al., 1998
; Twal et al., 2001
). Taken together, these findings suggest that the attachment of the extravillous cytotrophoblast to the endometrium during decidualization may be augmented by reduced cell motility induced by fibulin-1 and fibronectin, which is also up-regulated during decidualization (Zhu et al., 1992
). Furthermore, knockout studies (Kostka et al., 2001
) show that a fibulin-1 defect results in lethal changes in various small vessels. Since progesterone controls endometrial angiogenesis, and is critically involved in menstruation, fibulin-1 may also play a role in endometrial bleeding.
In conclusion, we have clearly demonstrated the induction of fibulin-1 mRNA and protein by progesterone during the decidualization of human ESCs, both in vivo and in vitro, which suggests that fibulin-1 is an important regulatory molecule in human endometrial cells for menstruation, implantation and placentation.
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Acknowledgements |
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Submitted on November 29, 2004; resubmitted on February 7, 2005; accepted on February 14, 2005.
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