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Departments of Obstetrics and Gynecology (S.L.Y.) and Statistics (P.L.S.), Columbia, Missouri 65212; Department of Obstetrics and Gynecology, University of North Carolina (B.A.L., M.A.F., W.R.M.), Chapel Hill, North Carolina 27599; The Permanente Medical Group, Inc. (M.J.M.), Sacramento, California 95815; Department of Obstetrics and Gynecology, University of California (M.J.M.), Davis, California 95817; and Department of Obstetrics and Gynecology, University of Texas Medical Branch (B.J.N.), Galveston, Texas 77555
Address all correspondence and requests for reprints to: Dr. Steven L. Young, Department of Obstetrics and Gynecology, University of Missouri, N625 HSC, 1 Hospital Drive, Columbia, Missouri 65212. E-mail: . youngst{at}health.missouri.edu
Abstract
Human endometrium expresses the critical complement component C3 in a cyclic fashion, with the highest expression in the secretory phase. As activated complement can kill cells, self or foreign, the secretory endometrial epithelium protects itself by concomitant expression of complement-protective proteins. The objectives of our present study were to describe the spatial and temporal regulation of the complement-protective protein decay-accelerating factor (DAF) in human endometrium and to identify local regulators of its expression. To describe the cyclic regulation of DAF, immunohistochemistry was performed using the IH4 monoclonal antibody on secretory phase endometrial biopsies taken from normal fertile volunteers in LH-timed cycles (n = 114). DAF expression in human endometrium was predominantly localized to the apical membrane of glandular and luminal epithelium. DAF expression, as assessed by histological scoring analysis, was minimal in the proliferative and early secretory phases and increased markedly on approximately day LH +7 (lumen) and LH +8 (glands). Maximal expression was seen in both glands and lumen by LH +8, and this persisted into menses. Using the RL95-2 endometrial epithelial cancer cell line as a model system, we next examined the cellular regulation of DAF. Treatment with E2 and progesterone, alone or in combination, had little effect on DAF expression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) treatment increased cell surface and total DAF protein, increasing the signal by 260% on flow cytometry and by 200% on Western blot analysis. Stimulation of DAF protein expression was dose dependent, with maximal expression seen at 1 ng/ml. The stimulatory effects of HB-EGF were also observed at the mRNA level. EGF had effects similar to those of HB-EGF on DAF mRNA and protein expression, suggesting that the HB-EGF effect was mediated at least in part by the Her1 EGF receptor subunit. These studies suggest that DAF expression in the midsecretory phase is stimulated by HB-EGF or other members of the EGF family and may function to protect the epithelial integrity of human endometrium in the face of increased complement expression.
THE IMMUNE SYSTEM is classically divided into two functional arms, innate and adaptive (1). The innate arm of immune defense includes the complement system, natural killer cells, epithelial integrity, and phagocytic cells. Innate immunity is rapid, does not depend on prior experience with a specific pathogen, and acts to stimulate adaptive immunity (2, 3). These properties determine a crucial role for the innate immune response at epithelial surfaces that contact the external environment, such as the endometrium.
Among epithelial surfaces, the endometrium is unique. Although it functions as a barrier to pathogen invasion, it must also tolerate the passage of foreign sperm and invasion by a semiforeign fetus. During the secretory phase, the presence of invading trophoblast and an intrinsic decrease in epithelial barrier integrity (4) probably provide an increased opportunity for pathogen infiltration. Our working hypothesis states that a weakening of the intrinsic endometrial barrier during the window of implantation requires a strengthening of the complement system to maintain a defense against pathogen invasion. This hypothesis provides a functional explanation of previous reports demonstrating increased expression of the critical complement component, C3, by the periimplantation endometrium in a number of species, including the human (5, 6).
Activated complement components can potentially opsonize and/or lyse any living cell, including pathogens, host cells, foreign gametes, and even the embryo. One mechanism by which the host can direct specificity of complement action is through the expression of complement-protective proteins found on the surface of many human cells (7). The normal human endometrial epithelium expresses two such complement-protective proteins, decay-accelerating factor (DAF) and membrane cofactor protein, both of which have been shown to prevent complement-mediated cell lysis in multiple cell types (7, 8, 9). Membrane cofactor protein is expressed at low levels throughout the menstrual cycle, whereas epithelial DAF expression is low during the proliferative phase and increases markedly in the secretory phase (5). As the degree of cellular protection against complement-mediated lysis is proportional to the level of DAF expression (10, 11), increased secretory phase DAF expression probably functions to protect the cells from increased complement levels, thereby preventing destruction of the host epithelium.
Endometrial epithelial DAF protein expression has been shown to be increased in the secretory phase (5, 12, 13, 14), but these studies had insufficient numbers of biopsies and cycle day coverage to provide full characterization of the temporal and spatial patterns of expression. We have previously shown that the secretory increases in DAF expression are absent in women with luteal phase defect, a disorder associated with suboptimal progesterone (P) levels, and were restored by P treatment (15). These studies suggested a vital role for P in the midsecretory increase of DAF expression. However, previous reports suggest that P receptor expression is greatly reduced in normal midsecretory and late secretory endometrial epithelia (16, 17). Therefore, many actions of P in the midsecretory phase are thought to be indirect, acting via P receptors in the adjacent stroma and involving the elaboration of paracrine factors (18, 19, 20). Among these factors is heparin-binding epidermal growth factor-like growth factor (HB-EGF), whose stromal and epithelial expression is increased in the midsecretory phase (21, 22, 23).
This paper describes the spatial and temporal changes in endometrial DAF expression using immunohistochemical analysis of LH-timed biopsies from fertile women. Based on the timing of epithelial DAF expression, we hypothesize that the secretory increase in DAF expression is dependent on increased expression of HB-EGF. To investigate this hypothesis, we tested the effects of HB-EGF treatment on DAF expression by RL95-2 cells, an in vitro human endometrial epithelial carcinoma cell line, known to contain functional steroid and EGF receptors (24, 25, 26).
Subjects and Methods
Human subjects
All human tissues were obtained using a protocol approved by the University of North Carolina committee for the protection of human subjects and in accordance with the Declaration of Helsinki. All subjects studied in the secretory phase were volunteers with a history of fertility. Additional samples were obtained during the proliferative phase from women at the time of tubal ligation. Secretory phase samples stained for DAF comprised 114 biopsies, each of which was performed based on a urinary LH (uLH) surge detection kit (Ovuquik, Quidel, San Diego, CA). At the time of the uLH surge, subjects were randomly assigned to return during the secretory phase (designated LH +1–14), as determined by a computer-generated randomization scheme. Samples were snap-frozen in liquid nitrogen immediately after biopsy. A small portion of each sample was fixed in formalin and stained with hematoxylin and eosin. Three pathologists, blinded to LH day, assigned a secretory day using standard criteria (27). If the LH day was discrepant from the pathologist assignment by more than 3 d, the sample was not included in the analysis. Proliferative phase endometrium was categorized based on time since last menstrual period and was confirmed as proliferative by the pathologist.
Immunohistochemistry
Immunohistochemistry was performed on 5-µm cryostat sections of endometrial biopsies using Vectastain Elite ABC kits (Vector Laboratories, Inc., Burlingame, CA). Diaminobenzidine (Sigma, St. Louis, MO) was used as the chromagen. After initial incubation with blocking antibody for 15 min at room temperature (1:100 dilution of nonimmune horse serum), primary antibody directed against DAF (IH4 hybridoma, provided by Dr. Douglas Lublin, Washington University, St. Louis, MO) was applied for 1 h. The primary antibody was used at a 1:100 dilution of medium taken from hybridoma culture (yield is usually 10–50 µg antibody/ml, giving a final antibody concentration of 0.1–0.5 µg antibody/ml). A PBS rinse was followed by secondary antibody consisting of biotinylated goat antimouse antibody for 30 min. After a PBS rinse, endogenous peroxidase was quenched with a 30-min incubation with 0.3% H2O2 in absolute ethanol, followed by a 30-min rehydration in PBS. Avidin-biotinylated horseradish peroxidase macromolecular complex was then incubated on the sections for 30 min before adding diaminobenzidine for 3 min to complete the reaction. Samples were subsequently washed in PBS and mounted. Negative controls included sections that were treated in the same manner with omission of the primary antibody as well as controls treated with a mouse IgG1 isotype control (eBioscience, San Diego, CA) at 0.5 µg antibody/ml. The resulting staining was evaluated on a Nikon microscope by a single blinded observer.
Assessment of staining intensity and distribution was made using the semiquantitative histological scoring (HSCORE) scoring system. HSCORE was calculated using the following equation: HSCORE = 
Pi (I + 1), where I is the intensity of staining with a value of 1, 2, or 3, (weak, moderate, or strong, respectively), and Pi is the percentage of stained epithelial cells for each intensity, varying from 0–100%. Low intraobserver and interobserver variabilities using the HSCORE technique in uterine tissues have been previously reported (28).
Cell culture
RL95-2 cells were originally obtained from the American Type Culture Collection (Manassas, VA), grown, and passaged in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% FBS (HyClone Laboratories, Inc., Logan, UT). For the experiments described, cells were grown in six-well tissue culture plates in 5% FBS until reaching 60–80% confluence. Cells were then incubated in DMEM supplemented with 5% heat-inactivated, charcoal-stripped FBS for at least 24 h before any subsequent treatment. E2 and P (Sigma) were made as stocks in 100% ethanol and diluted at least 1:10,000 in culture medium at the time of use. The cells were treated for the amount of time indicated with 10 ng/ml EGF, 10 ng/ml HB-EGF, 10-8 M E2, or 10-7 M P. In experiments where E2 and/or P treatments were used, all wells not containing E2 or P were treated with a 1:10,000 dilution of ethanol to control for any nonspecific effects of ethanol.
Flow cytometry
Cells were treated with hormones as described and collected from the plate by brief treatment with trypsin (0.25%)/EDTA (5.3 mM) (Life Technologies, Inc.). Trypsin was inhibited by FBS (10%), and the cells were washed twice with 10 ml PBS and counted. Cells (2 x 105) were resuspended in 80 µl PBS at 4 C, after which all subsequent steps were performed at 4 C in light shielded containers. Thirty microliters of CyChrome-conjugated antihuman DAF monoclonal antibody (BD PharMingen, San Diego, CA) were added and allowed to bind to the cells for 30 min. The amount of antibody used was determined empirically by titration as twice the saturating concentration. The cells were rinsed, diluted in 0.5 ml PBS, and analyzed on a FACScan flow cytometer (BD Immunocytometry Systems, San Jose, CA) using the software supplied by the manufacturer. Further analysis was performed using WinMidi software version 2.8 (Dr. Joseph Trotter, La Jolla, CA)
Western blot
RL95-2 cells were treated with EGF or HB-EGF using a 1:1000 stock in PBS with 0.5% BSA. Each well was washed once with PBS at 4 C, followed by addition of 500 µl RIPA lysis buffer [1 M Tris (pH 7.5), 5% Nonidet P-40, 10% sodium deoxycholate, and 1 M NaCl supplemented with 10 µl protease inhibitor cocktail (Sigma) and 5 µl 100 mM sodium orthovanadate]. The cellular material was recovered to microcentrifuge tubes by scraping and pipetting, followed by incubation on ice for 10 min. Insoluble components were removed by centrifugation at 16,000 x g for 10 min. An aliquot was saved for protein concentration determination using the bicinchinoic acid method according to the manufacturer’s instructions (Pierce Chemical Co., Rockford, IL), and the remainder was stored at -70 C for later analysis.
A 10% polyacrylamide-SDS gel with a 3.5% stacking layer was cast and run in a mini-Protean II device (Bio-Rad Laboratories, Inc., Hercules, CA). To each lane, 10 µg total protein in gel loading buffer [8% SDS, 0.25 M Tris-HCl (pH 6.8), 40% glycerol, and 0.04% bromophenol blue] were added, and electrophoresis was performed at 150 V for 1.5 h at 4 C. Transfer to a polyvinylidene difluoride membrane was performed using the mini-Transblot cell according to manufacturer’s instructions (Bio-Rad Laboratories, Inc.). The membrane was blocked with 1% BSA (wt/vol) and 4% nonfat dry milk in TBST buffer [10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween 20, and 1% BSA] for 1 h. The blot was incubated with primary antibody (IH4 monoclonal, gift from Dr. Denis Lublin, Washington University, St. Louis, MO) at a 1:1000 dilution in TBST overnight at 4 C. The membrane was washed with TBST six times for 5 min each time. The blot was then incubated in horseradish peroxidase-conjugated antimouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in the same buffer used for blocking and again washed six times as described above. The bands were then detected by exposure to ECL reagent (Amersham Pharmacia Biotech, Piscataway, NJ) and exposure to x-ray film (Biomax MR, Kodak, Rochester, NY). Numeric data were obtained by densitometric scanning analysis using AlphaEase software (Alpha-Innotech, San Leandro, CA).
We did not use a sulfhydryl reducing agent, because use of these reagents destroyed the capability for antibody recognition for all antibodies tried. Under the conditions described, the IH4 anti-DAF antibody stains a second, slightly smaller band, consistent with the studies of others (29). To confirm the identities of these bands, immunoblots were also stained with another anti-DAF antibody, IA10, yielding identical staining patterns. In addition, an extract from Chinese hamster ovary cells stably transfected with human DAF was used as a positive control.
Northern blot
mRNA was isolated by detergent lysis, followed by oligo(deoxythymidine)-cellulose binding and elution (FastTrack 2.0, Invitrogen, Carlsbad, CA). Northern blot was performed using a 1.8% formaldehyde-containing agarose gel, transferred by capillary blotting to nitrocellulose membrane (MSI, Westboro, MA), and hybridized with DAF probe generated by the random primer method using a linear DAF fragment from nucleotides 531–715 (30) generated by PCR from a DAF cDNA provided by Dr. D. Lublin (Washington University). The blot was then stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase riboprobe generated from a standard glyceraldehyde-3-phosphate dehydrogenase template using the MaxiScript kit (Ambion, Inc., Austin, TX).
Statistical analysis
HSCORE data were analyzed first using the Kruskal-Wallis ANOVA on ranks, as the data were nonparametric. Days 1–4 (the early secretory phase) were pooled and used as a control and then compared pairwise with each additional day using Dunn’s test. To support the hypothesis that the rise in luminal DAF expression occurred before the rise in glandular DAF, we compared the two curves using nonparametric methods with S-PLUS (Insightful, Seattle, WA). A smooth curve was computed for each dataset using the nonparametric data smoother LOESS (31). LOESS computes estimates at each day value by fitting a weighted least squares line to nearby data points lying within a window of the target day. The amount of data is controlled by the span parameter, typically stated as a fraction of the data. A span of 0.50 was used in Fig. 2C
, and 95% confidence bands for each curve were constructed. The curves were then compared using a novel nonparametric test based on previous parametric procedures for a one-sided test between two groups in the presence of a covariate (32, 33). In the parametric version, the combined data are regressed on covariates, and the residuals are analyzed using the Wilcoxon rank-sum test. We have extended the method by using LOESS to smooth the combined data in place of standard regression. No additional assumptions are made regarding the distributions of the data, except for independent observations. In particular, it is not necessary to assume equal variances. This novel analysis method was used to test the hypothesis that the mean HSCORE values for luminal and glandular cells are equal on each day against the hypotheses that the lumen HSCORE means are greater than or equal to the gland means with strict inequality on at least 1 d.
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Results
To determine the spatial and temporal changes in DAF expression in human secretory endometrium, we performed immunohistochemical staining for DAF in uLH-timed biopsy material from fertile, cycling women. Figure 1 demonstrates individual immunohistochemical staining of endometrial biopsies for DAF. Minimal DAF staining was found in the proliferative and early secretory phases, with an increase in the midsecretory phase that was sustained through the late secretory phase. Immunostaining for DAF was localized predominantly to the glandular and luminal epithelia, with some staining of blood vessel epithelium and minimal staining of stromal cells. Interestingly, staining of the endometrial epithelium was predominantly apical and appeared to intensify as the cycle progressed.
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represents a semiquantitative assessment of endometrial epithelial DAF expression in 114 endometrial biopsies obtained throughout the secretory phase using the HSCORE method (28). Although some variability was noted between samples, luminal DAF expression appeared to increase on or before LH +7 (Fig. 2A), whereas glandular expression appeared to increase slightly later, on or before LH +8 (Fig. 2B). In both luminal and glandular epithelia, maximal expression was achieved by LH +8 and remained at that level until menstruation. To support the hypothesis that luminal DAF expression precedes that in the glands, we first computed a smooth curve and 95% confidence interval of the mean for lumen and glands separately using the nonparametric data smoother LOESS (Fig. 2C). The confidence intervals did not overlap on d 5 and 6, suggesting that the rise in HSCORE occurs in luminal cells before that in glandular cells. These curves were further analyzed using a new nonparametric test described in Subjects and Methods. The hypothesis tested is that the mean HSCOREs obtained for luminal and glandular cells are equal on each day against the hypotheses that the lumen HSCORE means are greater than or equal to the gland means with strict inequality on at least 1 d. With a span of 0.5, the test was significant with P = 0.004. The result was relatively insensitive to the choice of span, with P < 0.009 for span choices between 0.3 and 0.7. Thus, this analysis supports the hypothesis that increased luminal expression precedes increased glandular expression.
To study the regulation of DAF we used the endometrial cell line, RL95-2. These cells maintain both EGF receptor and hormone responsiveness (24, 25, 26). As DAF exerts its protective effects on the cell surface, we first assessed the effects of HB-EGF treatment on expression of cell surface DAF using flow cytometry (Fig. 3). Typical histograms (Fig. 3A) demonstrate a more than 10-fold difference in signal intensity between an isotype control antibody and the anti-DAF antibody, indicating appropriate specificity of the anti-DAF antibody. The amount of DAF immunofluorescence was increased by a 24-h treatment with 10 ng/ml HB-EGF (Fig. 3A). In addition, we measured the response to 10 ng/ml EGF (Fig. 3A), which can use the same receptor as HB-EGF. The responses to EGF, HB-EGF, E2, and E2 plus P are quantitated in Fig. 3B. There were 260% and 220% increases in cell surface DAF staining after treatment with HB-EGF and EGF, respectively. E2 treatment had no demonstrable effect on DAF expression. P treatment for 24 h in either the presence or absence of estrogen appeared to cause a small (12%) reduction in DAF staining that did not achieve statistical significance in repeated studies. Because some steroid effects are known to be maximal beyond 24 h, we treated the cells with E2 and/or P for 2–5 d, which also did not result in any significant changes in DAF expression. In addition, treatment with steroid hormones did not interfere with the stimulation by EGF or HB-EGF (data not shown).
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. On repeated experiments it appeared that maximal stimulation of DAF expression by either HB-EGF or EGF occurred at doses between 1 and 5 ng/ml.
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, demonstrated that RL95-2 cells produce both 2.2- and 1.5-kb forms of DAF transcript, as reported in other cell types (34). In addition, there was a larger band apparent at the 4 h point at about 2.5 kb that has not been previously noted and may represent incompletely spliced RNA. Treatment with either EGF or HB-EGF increased both mRNA species within 4 h, after which there was a slow decline (Fig. 5A). Densitometric scanning revealed an approximately 88% increase in the combined intensity of both bands after treatment with EGF, whereas treatment with HB-EGF caused a 73% increase. The blot was stripped and reprobed with the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a control for differences in loading and transfer between lanes. Densitometric scanning demonstrated less than a 5% difference between lanes using the GAPDH probe. Other experiments (Fig. 5B) demonstrate increases in DAF with as little as 30 min of HB-EGF treatment and peak expression by 2 h. The apparent small change in migration of the DAF band in Fig. 3B appears to be secondary to "frowning" of the gel.
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These studies have demonstrated spatial and temporal regulation of DAF expression in the human endometrial epithelium. A large increase in expression was observed in cycling fertile women 7–8 d after the uLH surge, with increased expression persisting throughout the secretory phase. We also observed evidence for spatial regulation, with expression predominantly on the apical cellular membrane of the epithelium. Our findings are consistent with previous reports indicating increased secretory phase expression of DAF (5, 15). In contrast to previous studies, the biopsies used in this study were obtained from normal fertile women, timed to the uLH surge, included many more subjects, and examined the entire secretory phase.
The timing of increased DAF expression is coincident with peak P levels, consistent with our previous data showing that P treatment of patients with luteal phase defect was associated with a return of normal DAF expression (12). Both the lack of P stimulation in our in vitro model and the marked decline in P receptor expression by the endometrial epithelium in vivo (16, 17) suggest that P acts indirectly to stimulate DAF expression during the midsecretory period. As stromal cells maintain P receptor and retain P responsiveness, it is likely that the increase in epithelial DAF expression in the midsecretory phase is caused by P-induced paracrine factors produced in the stroma.
One potential paracrine/autocrine factor whose peak expression coincides with the increase in DAF expression is HB-EGF (21, 22, 23). To assess the potential for HB-EGF stimulation of DAF expression, we measured the effects of HB-EGF treatment of RL95-2 cells. HB-EGF stimulated the expression of cell surface and total DAF protein as well as DAF mRNA, supporting the hypothesis that HB-EGF is an important physiological regulator of endometrial DAF expression.
Understanding the physiological role of HB-EGF and other EGF family members in the endometrium has been complicated by the fact that there are multiple EGF receptor ligands (e.g. EGF, HB-EGF, amphiregulin, betacellulin, and TGF
) that can bind multiple receptor subunits (ErbB1, ErbB2, ErbB3, and ErbB4) (35). To better understand the mechanism of HB-EGF action, we treated RL95-2 cells with EGF, which showed effects similar to those of HB-EGF on DAF expression. Therefore, although previous reports on endometrial HB-EGF have emphasized the potential effects on the embryo (22), our studies suggest a potential effect on the endometrium. The physiological importance of apical DAF expression in human endometrium remains unclear, but the critical role of complement protection at the fetal-maternal interface in mice has recently been demonstrated (36).
The finding that DAF expression is regulated by the EGF family of ligands is also of interest, because previous reports describing DAF regulation have focused almost entirely on cytokines (37, 38, 39, 40), with a single study suggesting regulation by nerve growth factor (41). Our data present the first evidence of DAF regulation by two additional growth factors, EGF and HB-EGF. Although there appears to be a difference in dose response between EGF and HB-EGF (a decline at higher concentrations of EGF), this may be because of differences in aggregation in solution, interactions at a single receptor type, or action at overlapping subsets of EGF receptor types, especially at higher doses. It is important to note that the near ubiquity of the EGF ligands and receptors coupled with our findings suggest that DAF regulation by EGF family ligands represents a physiologically important mechanism in other reproductive and nonreproductive organs.
In conclusion, our data demonstrate the following. 1) DAF expression increases markedly on cycle days LH +7 (lumen) to LH +8 (glands), almost exclusively at the apical aspect of the epithelium. 2) Both HB-EGF and EGF can stimulate DAF mRNA and protein expression. 3) The temporal relationship between changes in HB-EGF and DAF expression in vivo and demonstration of effects in vitro suggest that HB-EGF is an important physiological stimulus for the midsecretory increase in DAF expression. Considering the well described protective functions of DAF, HB-EGF may be an important regulator of innate immunity and epithelial integrity in the secretory endometrium.
Acknowledgments
We thank Wen-Ru Zhang for her excellent technical assistance with the Northern blots, Western blots, and flow cytometry; Jining Zhang and Terri Lyddon for their excellent technical assistance with the immunohistochemistry; and the University of Texas Medical Branch flow cytometry core laboratory and its manager, Mark Griffin, for invaluable assistance with flow cytometry.
Footnotes
This work was supported in part by the NICHHD/NIH through Cooperative Agreement U54-HD-35041 (to B.A.L.) as part of the Specialized Cooperative Centers Program in Reproduction Research, the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation Grant HD-34824 (to B.A.L.), and NIH Grant NIDDK-42029 (to B.J.N.). S.Y. completed most of this work as a scholar at the University of Texas Medical Branch Women’s Reproductive Health Research Career Development Center of Excellence (NIH Grant 5K12-HD-01269-2). Presented at the Annual Meeting of the Society for Gynecologic Investigation, Chicago, Illinois, 2000.
Abbreviations: DAF, Decay-accelerating factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HB-EGF, heparin-binding epidermal growth factor-like growth factor; HSCORE, histological scoring; P, progesterone; uLH, urinary LH; TBST, 10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween 20, and 1% BSA.
Received April 27, 2001.
Accepted December 6, 2001.
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) so that control levels are identical to those with HB-EGF (
).