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Department of Obstetrics and Gynecology (H.-Q.W., K.Ta., Y.N.) and Molecular Neuroscience Research Center, (M.N.), Otsu 520-2192, Shiga, Japan; and Institute for Enzyme Research, University of Tokushima (K.Ts.), Kuramoto, Tokushima, 770-8503 Japan
Address all correspondence and requests for reprints to: Koichi Takebayashi, M.D., Department of Obstetrics and Gynecology, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu 520-2192, Japan. E-mail: ktakebay{at}belle.shiga-med.ac.jp.
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Abstract |
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Introduction |
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Human endometrium produces bioactive and immunoreactive inhibins and activins, suggesting possible roles for these hormones/growth factors in the physiology of the endometrium (1, 2, 3, 4, 5). A recent study revealed that activin enhances the decidual reaction in vitro (6). Follistatin, a secreted glycoprotein, is able to neutralize the various activities of activin by forming an inactive complex with it (7) and is expressed in endometrial epithelial cells of the secretory stage of the menstrual cycle and decidual cells of early pregnancy (8).
Follistatin-related gene (FLRG), originally identified from the molecular study of a t (11, 19) (q13;p13) translocation observed in a case of B cell chronic lymphocytic leukemia, encodes a secreted glycoprotein highly homologous with follistatin (9). As an extracellular regulatory protein, like follistatin, FLRG protein binds activins and bone morphogenetic proteins (BMPs) and controls their functions extracellularly (10, 11, 12, 13, 14). FLRG is expressed in various types of human tissues, including placenta, heart, aorta, testis, adrenal gland, and uterus (10, 13). In a hepatoma cell line (HepG2) and human bone marrow stromal cells, FLRG expression has been shown to be up-regulated by TGFß (12, 15).
The studies performed to date have strongly suggested that activins, inhibins, follistatin, and FLRG protein could interact with each other and play important roles through autocrine/paracrine pathways in the uterus. To clarify the regulation of FLRG expression in human endometrium and further the understanding of uterine physiology, tissue and intracellular localization of FLRG protein and mRNA expression were evaluated in endometrial specimens at different stages of the menstrual cycle and in decidua. Furthermore, using an in vitro decidualization model (16), we investigated the effects of two ovarian hormones on FLRG expression in cultured human endometrial stromal cells.
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Materials and Methods |
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This study was approved by the institutional review board at Shiga University of Medical Science. Written informed consent was obtained from every patient.
Endometrial tissues were obtained at hysterectomy or as biopsy specimens from normally cycling premenopausal women (n = 56: early-mid proliferative phase, n = 22; early secretory phase, n = 9; mid-late secretory phase, n = 25), aged 32–46 yr, who had received no hormonal treatment and underwent surgery for nonendometrial abnormalities, such as myoma uteri or cervical carcinoma in situ. A portion of each endometrial tissue was examined histologically and dated according to the criteria of Noyes et al. (17).
Decidual tissues were obtained at elective termination of normal pregnancy from women (n = 32) at 6–8 wk gestation. Decidua without trophoblast was confirmed by histological examination of hematoxylin- and eosin-stained sections. All samples were washed several times with Dulbecco’s PBS (Nissui Pharmaceutical Co., Tokyo, Japan) to remove blood clots. A part of each sample was fixed for 4–6 h at 4 C in 4% paraformaldehyde (Nacalai tesque, Kyoto, Japan) for immunohistochemical analysis. The remaining tissue was used for cell culture.
Human endometrial stromal cells (ESC) were isolated and cultured as previously described (16). Briefly, tissue samples were minced into small pieces of less than 1 mm3. These tissue pieces were then incubated for 1 h at 37 C in a humidified atmosphere of 5% CO2 in air in DMEM (phenol red-free; Life Technologies, Inc., Grand Island, NY) containing 0.2% collagenase (from Clostridium histolyticum; Wako Pure Chemical Co., Osaka, Japan) and 0.005% deoxyribonuclease I (type IV, from bovine pancreas; Sigma-Aldrich Corp., St. Louis, MO), with gentle pipetting every 15 min. After digestion, the cell suspension was left in an upright position for 5 min. Then the supernatant, the stromal cell-rich fraction, was transferred onto 40-µm pore size nylon mesh (BD Biosciences, Franklin Lakes, NJ) and centrifuged for 10 min. The purity of stromal cells obtained by this method was usually greater than 95%, as determined by immunohistochemical staining against vimentin (stromal cell marker) and cytokeratin (epithelial cell marker). The purified stromal cells were washed three times, and the number of viable cells was counted by trypan blue dye exclusion. One million viable cells were inoculated into each well of six-well plates (BD Biosciences).
Cells were cultured at 37 C in a humidified atmosphere of 5% CO2 in air with 4 ml DMEM supplemented with 10% charcoal-stripped fetal bovine serum (Life Technologies, Inc.), 50 IU/ml penicillin, 50 µg/ml streptomycin (Sigma-Aldrich Corp.), and with or without 17ß-estradiol (E2; Sigma-Aldrich Corp.) and/or progesterone (P; 4-pregnene-3,20-dione; Sigma-Aldrich Corp.). The morphological transformation of ESC and the elevation of PRL in the supernatant of culture medium were used as markers for in vitro decidualization (16). PRL in each culture medium was measured by RIA (Daiichi Radioisotope Laboratory, Tokyo, Japan). The detection limit of this assay was 0.3 ng/ml, and the intra- and interassay coefficients of variation were 1.9–7.1% and 1.6–3.6%, respectively. Unless indicated otherwise, medium was renewed every 2 d during the culture period. Each experimental set-up was repeated on at least three occasions using endometrial stromal cells obtained from different patients.
Immunohistochemistry
Endometrial specimens were fixed in 4% (wt/vol) paraformaldehyde, followed by sequential sucrose dehydration and OCT (Tissue-Tek, Miles, Elkhart, IN,) embedding. Serial sections (8 µm) were made by a cryostat and were mounted on 3-aminopropyltriethoxysilane-coated glass slides.
A monoclonal antibody against the COOH-terminal region of human FLRG protein was provided by Kyowa Hakko Kogyo Co. Ltd. (Machida, Tokyo, Japan) and was used to identify FLRG protein localization in various menstrual stages of endometrium and decidua by standard immunohistochemical techniques, using the avidin-biotin immunoperoxidase method. To verify the quality of the tissue, every 10th sample was stained with hematoxylin and eosin. Endogenous peroxidase enzyme activity was quenched with 0.3% (vol/vol) hydrogen peroxide in methanol at room temperature for 30 min. Nonspecific binding sites were blocked using 2.5% (vol/vol) normal goat serum in Ca2+ and Mg2+ free PBS [PBS (-)] for 10 min at room temperature. Sections were incubated overnight at 4 C with the primary antibody diluted to 1 µg/ml in PBS (-) containing 1.5% (vol/vol) normal goat serum as a blocker. Sections were washed three times for 5 min each time in PBS (-) and incubated for 10 min at room temperature with biotinylated universal secondary antibody diluted in PBS (-) containing normal blocking serum as a blocker. Sections were rinsed three times for 5 min each time in PBS (-) and incubated with avidin-biotin-peroxidase complex at room temperature for 5 min. Finally, antigenic sites were visualized by 3,3-diaminobenzidine in PBS (-), pH 7.5, containing 0.003% (vol/vol) H2O2, giving rise to a brown product. After light counterstaining with Mayer’s hematoxylin, slides were dehydrated in an ascending ethanol series, cleared in xylene, and mounted. Immunoneutralization was performed by incubating sections with preneutralized primary antibody with a 10-fold molar excess of a synthetic immunopeptide provided by Kyowa Hakko Kogyo Co. Ltd. (Machida, Tokyo, Japan).
Laser scanning confocal microscopy
The same initial treatment was used for the immunostaining for confocal microscopy (MRC-600 Laser Scanning Confocal Imaging System, Bio-Rad Laboratories, Hemel Hempstead, UK) as for immunohistochemistry with the following exceptions. The peroxidase treatment was omitted. Immune complexes were visualized with Alexa Fluor 596 goat antimouse IgG (Molecular Probes, Inc., Eugene, OR). Nuclei were counterstained with 4',6-diamidino-2-phenylindole, dihydrochloride (Nacalai tesque, Kyoto, Japan).
Western blot analysis
Endometrial blood vessels were removed mechanically under microscopy, and extensively washed with PBS (-). Afterward the samples were chopped, further washed extensively with PBS (-), and homogenized. Tissue proteins were extracted in a buffer solution containing 0.5% Triton X-100, 10 mmol/liter HEPES (pH 7.4), 150 mmol/liter NaCl, 2 mmol/liter EGTA, 2 mmol/liter EDTA, and a complete protease inhibitor cocktail. After centrifugation for 30 min at 15,000 x g, the concentration of protein in a soluble fraction was measured by the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instruction. Equal amounts (50 µg) of proteins were electrophoresed through a 12% sodium dodecyl sulfate-polyacrylamide gel and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membrane was blocked with 5% nonfat milk and incubated overnight with an antihuman FLRG monoclonal antibody. Immunological complexes were revealed with a horseradish peroxidase-conjugated goat antimouse IgG (Pierce Chemical Co.). The proteins were detected using an enhanced chemiluminescence Western blotting kit (Amersham Pharmacia Biotech, Uppsala, Sweden).
RT-PCR
Total RNA was extracted using TRIzol (Life Technologies, Inc.) according to the manufacturer’s instructions, quantified by measuring absorbance at 260 nm, and stored at -80 C until assay. Five micrograms of total RNA were converted to cDNA with 0.5 µg oligo(deoxythymidine)12–18 primers (Life Technologies, Inc.) using Superscript II reverse transcriptase (Life Technologies, Inc.) in a final volume of 20 µl. The RT was carried out for 60 min at 42 C. The blank for each RT reaction consisted of all of the reagents with water substituted for the reverse transcriptase. Products were stored at -20 C until the subsequent PCR.
One microliter of RT reaction product was used in PCR. Elongation factor-1
(EF-1) signal was measured in all RNA samples without RT to detect genomic contamination. Aliquots of the RT products were subjected to PCR using LA Taq polymerase (Takara) to amplify FLRG and Ex Taq polymerase (Takara) to amplify EF-1. An aliquot of each PCR solution was fractionated by electrophoresis in a 1.5% agarose gel. Gels were stained with ethidium bromide, destained, and photographed.
All experiments were performed at least three times with similar results. In each experiment the PCR products were directly sequenced using the Rhodamine Terminator Cycle sequencing ready reaction kit (PE Applied Biosystems, Warrington, UK) to confirm the accuracy of the PCR products, as previously described (18).
Oligonucleotide primers for RT-PCR
Two primers used for FLRG amplification were as follows. The forward primer was 5'-TGTGCCTCCGGCAACATTGAC-3', and the reverse primer was 5'-TAGGTGACGTTGTTGTTGCC-3'. The expected size of the amplified fragment was 470 bp.
EF-1 message was amplified as an internal control molecule. The forward primer was 5'-CGTGGTATCACCATTGATATCTCC-3', and the reverse primer was 5'-TTGAGAACACCAGTCTCCACTC-3'. The expected size of the amplified fragment was 614 bp.
Competitive PCR
Using an internal standard cDNA for competitive PCR, regulation of FLRG expression in cultured human stromal cells was investigated as previously described (19, 20, 21).
A competitive cDNA fragment was constructed by deletion of an 89-bp fragment from the FLRG cDNA in pBS SK- plasmid (10) as illustrated representatively in Fig. 1
. After digestion with BssHII, the pBS SK- containing 89-bp BssHII-restriction site-directed deletion of FLRG cDNA was self-ligated and subcloned. The amplified plasmid was linearized with XbaI digestion and used as an internal control in competitive PCR. The linear portion of the amplification relationship between target cDNA and internal control cDNA was determined. For that purpose, a fixed amount of cDNA derived from ESC treated with E2 and P was mixed with a series of 10-fold dilutions of the internal control cDNA, then the target and the internal control cDNAs were coamplified by PCR using the primers described above. Competitive PCR was performed using LA Taq DNA polymerase under the following conditions: 30 cycles of denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec, and extension at 72 C for 2 min. Aliquots of PCR products were electrophoresed on 8% polyacrylamide gel, visualized after ethidium bromide staining, photographed, and scanned (Fig. 2A). The relative integrated density of each band was digitized by multiplying the absorbance of the surface area (Luminator Imaginator II, Funakoshi Co., Tokyo, Japan). Then the ratios of the densitometric readings of the amplified target cDNA and internal control cDNA were plotted on the ordinate against the serial 10-fold dilutions of internal control cDNA on the abscissa (Fig. 2B). After establishing the working ranges in which linear relationship existed, a 10-6 µg/µl concentration of the internal control cDNA was selected. For competitive PCR reactions, 1 µl of internal control samples was added to l µl of each cDNA sample. The resultant PCR products were quantified by densitometric scanning as described above. The abilities of sex steroid hormones to modulate FLRG mRNA expression were measured by calculating the ratio of target cDNA to internal standard cDNA.
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All results shown are the mean ± SEM of at least three separate experiments, with triplicate determinations for each treatment. Differences among groups were analyzed using ANOVA, followed by Fisher’s least significant difference multiple comparison test; P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FLRG expression in human endometrium was initially assessed by RT-PCR in homogenates of specimens in various stages of the menstrual cycle (n = 56) and at 6–8 wk gestation (n = 32). A single band corresponding in size to the FLRG product was obtained in all the samples examined (data not shown). The identities of the PCR products were confirmed by determination of DNA nucleotide sequences (data not shown). There was patient to patient variation in the intensities of FLRG mRNA signals in endometrium; however, a trend toward increased expression of FLRG was observed in secretory endometrium compared with proliferative endometrium, and further increased expression was observed in first trimester decidua (Fig. 3). In all experiments, negative controls yielded no detectable products, indicating that 1) all reagents were free of target sequence contamination; and 2) the RT-PCR products do not come from contaminating genomic DNA.
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Western blot analysis for detection of FLRG protein revealed the single band of approximately 36 kDa from all samples examined (Fig. 4A). The relative amount of FLRG protein was significantly higher in the secretory phase than in the proliferative phase of the menstrual cycle and reached the maximum level in the decidua of early pregnancy (Fig. 4B).
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Tissue distribution of FLRG protein in human endometrium was analyzed by immunohistochemistry. In the proliferative phase of the menstrual cycle, only epithelial cells were immunoreactive for FLRG protein, and stromal cells showed no staining (Fig. 5A). In the secretory phase, however, the expression of FLRG protein was also detected in some of the stromal cells (Fig. 5B).
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). Either substitution of primary antibody with goat normal IgG or preincubation of primary antibody with the FLRG immunopeptide completely abrogates immunoreactivity (Fig. 5, D–F). Intracellular localization of FLRG protein in endometrium during menstrual cycle and in decidua
In the proliferative phase of the menstrual cycle, FLRG protein was predominantly and homogeneously distributed to the cytoplasm of epithelial cells (Fig. 6, A and B). In the secretory phase (Fig. 6, C and D) and in early pregnancy (Fig. 6, E and F), FLRG protein was also strongly detected in the nucleus of most endometrial stromal cells, although some stromal cells demonstrated positive cytoplasmic and negative nuclear staining.
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To investigate whether sex steroid hormones induce FLRG expression in human ESC more directly, we measured the FLRG mRNA expression levels of cultured ESC using competitive PCR. Firstly, we established a linear relationship between the expression of target FLRG and the internal control as described above (Fig. 2, A and B). As a result, we selected a 10-6 µg/µl concentration of internal control to perform the competitive reactions. To determine the effects of E2 and P on FLRG expression in ESC, P was added to cells obtained in various stages of the cycle at concentrations of 10-11–10-5 mol/liter for 14 d in absence or presence of 10-8 mol/liter E2. P stimulated FLRG expression in a dose-dependent manner, reaching a maximum level at 10-7 mol/liter (Fig. 7A) accompanied by the release of PRL (Fig. 7B). Then, in the presence of 10-7 mol/liter P, E2 was added to the cells at concentrations of 10-12–10-6 mol/liter for 14 d. Under the fixed amount of 10-7 mol/liter P, 10-8 mol/liter E2 had the maximum effect on FLRG expression (Fig. 7C) with increasing PRL secretion (Fig. 7D). Therefore, the optimal concentrations of E2 and P for FLRG expression were determined to be 10-8 and 10-7 mol/liter, respectively. These values are within the physiological limits of blood serum concentrations during normal pregnancy, and these observations were consistent with our previous report (16). Moreover, in the presence of 10-8 mol/liter E2 plus 10-7 mol/liter P (E2/P), FLRG expression increased time-dependently until 14 d of culture and reached the maximum levels of FLRG expression (Fig. 8A) and PRL secretion (Fig. 8B) at 14 d of culture.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human endometrium expresses mRNAs for inhibin, activin, and follistatin, and the presence of both immunoreactive and bioactive proteins has been demonstrated (1, 2, 3, 25, 26, 27, 28, 29). Although FLRG and follistatin are structurally and functionally related, several differences have been shown between follistatin and FLRG. The expression of follistatin mRNA is rather ubiquitous compared with that of FLRG mRNA (10, 13), and follistatin and FLRG transcriptions are differently regulated by protein kinases A and C (11, 30). Moreover, FLRG protein, unlike follistatin, was localized to the nucleus in many cell types and was nearly 100-fold less potent than follistatin in its ability to neutralize endogenous activin (13, 14).
In this study the expression level of FLRG protein in the human endometrium was found to be higher in the secretory phase and early pregnancy than in the proliferative phase because of the additional induction of FLRG expression in ESC. In the secretory phase, follistatin was predominantly and heterogeneously expressed in the glandular epithelium (8), whereas FLRG was also expressed in stromal cells. Most stromal cells in the secretory phase and most decidual cells showed nuclear localization of FLRG protein; however, some were negative for nuclear staining. This heterogeneously expressed pattern may result either from heterogeneity of stromal cells per se or from temporally different developing process of decidualization. Taken together, these results raise the possibility that follistatin and FLRG have distinct regulatory pathways in human endometrium.
Finally, we showed that E2 and P, but not either alone, greatly induced FLRG expression through in vitro decidualization of ESC. These results suggest that FLRG is regulated at least in part by ovarian steroid hormones in the differentiating ESC and might be produced differently in endometrial epithelial cells and ESC.
In conclusion, we present clear evidence that human endometrial cells express FLRG, and ovarian steroid hormones modulate its synthesis in vitro. Further investigation will uncover the complex mechanisms of human endometrial differentiation and enhance our understanding of the control of reproduction.
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Acknowledgments |
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Footnotes |
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Abbreviations: BMP, Bone morphogenetic protein; E2, 17ß-estradiol; EF-1, elongation factor-1; ESC, endometrial stromal cells; FLRG, follistatin-related gene; P, progesterone; PBS (-), Ca2+ and Mg2+ free PBS.
Received November 8, 2002.
Accepted May 28, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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