| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Studies |
Department of Obstetrics and Gynecology, Division of Human Reproduction and Infertility, University of North Carolina, Chapel Hill, North Carolina 27599-7570
Address all correspondence and requests for reprints to: Dr. Bruce A. Lessey, Department of Obstetrics and Gynecology, CB #7570, Old Clinic Building, University of North Carolina, Chapel Hill, North Carolina 27599-7570.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
vß3
integrin cell adhesion molecule appears to be regulated in the human
endometrium, appearing on postovulatory days 5–6, corresponding to the
time of initial embryo attachment. This integrin has been extensively
studied as a potential marker of endometrial receptivity and is
aberrantly expressed in the endometrial epithelium of some infertile
women. Ishikawa cells are a well differentiated endometrial
adenocarcinoma cell line that maintain functional estrogen and
progesterone receptors and are a useful model to study steroid-mediated
events in human endometrial epithelium. This cell line expresses most
of the normal endometrial epithelial integrins, including the
vß3 vitronectin
receptor. The regulation of this integrin was studied with fluorescence
immunocytochemistry, flow cytometry, and Northern blot analysis.
Estrogen with or without progesterone treatment down-regulates
vß3 in this cell line.
Several growth factors, including epidermal growth factor and the
closely related transforming growth factor- significantly increase
the expression of this integrin. We conclude that endometrial
differentiation is influenced by both steroid hormones and growth
factors. The vß3
integrin appears to be an excellent marker to study the molecular
events leading to the establishment of uterine receptivity and
successful implantation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Certain integrins, members of a family of cell adhesion molecules, have
emerged as effective markers of endometrial receptivity in the human
(10, 11). These heterodimeric glycoproteins are present on the surface
of virtually all cells and participate in cell-cell and cell-substratum
interactions with the extracellular matrix (12, 13). Each integrin
consists of an -subunit and a ß-subunit (14). We (10, 15) and
others (11) recently described the patterns of integrin expression in
the normal cycling endometrium. Three cycle-specific integrins have now
been described, the 1ß1 collagen receptor,
the 4ß1 fibronectin receptor, and the
vß3 vitronectin
receptor, all coexpressed only during the suspected window of
implantation (15). Interestingly,
vß3 is not expressed
in the endometrium of some women with minimal and mild forms of
endometriosis (16), luteal phase deficiency (10, 17), communicating
hydrosalpinx (18), recurrent pregnancy loss (19), and unexplained
infertility (20).
The cycle-specific patterns of integrin expression in the endometrium
suggest that these molecules are hormonally regulated (10, 11, 15).
During the midsecretory phase of the endometrial cycle, when
implantation presumably occurs, circulating levels of estrogen (E) and
progesterone (P) are high, resulting in an abrupt decline in E and P
receptors (ER and PR) in the endometrial epithelium (21, 22). Recently,
we have shown that this down-regulation of PR closely correlates with
the rise in endometrial
vß3 expression and
that in women with documented luteal phase deficiency, delayed
endometrial maturation is associated with persistence of epithelial PR
and absent vß3
expression (17). This reciprocal relationship between PR and the
vß3 integrin suggests
that the expression of this integrin and, by inference, the onset of
uterine receptivity, may normally be inhibited by E and P, appearing
only after the supply of epithelial receptor for these steroids is
exhausted. Evidence suggests that certain growth factors, in particular
epidermal growth factor (EGF) and related species, may also play a role
in rendering the endometrium receptive to embryo implantation (23, 24).
Few models exist for studies of human endometrium in vitro.
Ishikawa cells represent a well differentiated endometrial
adenocarcinoma cell line (25) that is well suited for studies of
hormonally regulated events in human endometrial epithelium (26, 27, 28, 29).
These cells are unique, in that they exhibit functional ER (26, 27) and
estrogen-induced functional PR (28). Patterns of integrin expression in
Ishikawa cells are remarkably similar to those observed in normal
endometrial epithelium. As in native endometrium,
2ß1,
3ß1, and
6ß4 expression is constitutive, whereas
1ß1 and
vß3 are sensitive to
hormonal regulation (29). Consequently, we employed this model system
in studies designed to further investigate the mechanisms that regulate
expression of the vß3
integrin, which is now gaining acceptance as a valid molecular marker
of endometrial receptivity in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Ishikawa cells were cultured to confluence in
150-cm2 flasks (Costar, Cambridge, MA) containing
DMEM-Ham’s F-12 supplemented with charcoal-stripped FCS (pH 7.2), 200
mmol/L L-glutamine, and penicillin/streptomycin at 37 C in
95% air-5% CO2. Reagents were obtained from Sigma
Chemical Co. (St. Louis, MO) except where otherwise specified. Cells
were cultured in the presence or the absence of 10-8 mol/L
17ß-estradiol (E) and/or 10-6 mol/L P. Hormones were
drawn from a 1000-fold concentrated stock in 100% ethanol (ethanol),
maintaining equal concentrations of ethanol in control flasks.
Steroid-treated and untreated cells were also cultured in the presence
of recombinant human EGF (0.1–10 ng/mL), recombinant basic fibroblast
growth factor (bFGF; 10 ng/mL), recombinant insulin-like growth factor
II (IGF-II; 0.5 ng/mL), recombinant human interleukin-1 (IL-1)
and IL-1ß (0.5 and 0.2 ng/mL, respectively), macrophage
colony-stimulating factor (m-CSF; 20 U/mL), recombinant
platelet-derived growth factor (PDGF-AA, -AB, and -BB; 10 ng/mL),
recombinant human transforming growth factor- (TGF; 5 ng/mL),
TGFß (0.5 ng/mL), and recombinant human tumor necrosis factor-
(TNF; 5 ng/mL) for a period of 6 days. In separate experiments, the
influence of dosage and time in culture were investigated.
Immunocytochemistry and flow cytometry
Fluorescence immunocytochemistry was performed using
formalin-fixed Ishikawa cells that had been cultured on coverslips,
using a Vectastain kit (Vector Laboratories, Burlingame, CA). After
initial incubation with blocking antibody for 15 min at room
temperature (1:100 dilution of nonimmune mouse serum), primary antibody
was applied for 1 h. Monoclonal antibodies (mAbs) directed against
the ß3-subunit (AP3 and SSA6) were provided by
Drs. Peter Newman (Milwaukee, WI) and James Hoxie (University of
Pennsylvania, Philadelphia, PA). Both have been characterized
previously (10, 15) and can detect intact
vß3 in
situ. After a 5-min wash in phosphate-buffered saline (PBS; pH
7.2–7.4), biotinylated goat antimouse IgG conjugated to fluorescein
isothiocyanate (1:100 dilution) was applied to the coverslips. After a
30-min incubation at room temperature, coverslips were rinsed three
times with PBS and mounted. Photomicrographs were prepared using Kodak
(Rochester, NY) TMZ 3200 ASA film in a Nikon Optiphot fluorescence
microscope (Melville, NY).
In preparation for flow cytometry, Ishikawa cells were first detached from culture flasks by light trypsin-ethylenediamine tetraacetate digestion for 5 min (Life Technologies, Grand Island, NY), then neutralized with an equal volume of medium containing 5% FCS and centrifuged at 300 x g for 10 min. The supernatant was discarded, and cells were resuspended in a volume of PBS to yield a concentration of 100,000 cells/mL. A 500-µL aliquot of cell suspension was transferred to individual polypropylene tubes, pelleted, resuspended in 300 µL PBS-4% BSA containing primary mouse antihuman mAb (ß3; 1:100), and incubated for 60 min at 4 C on a shaker. Tubes were then centrifuged for 10 min at 300 x g, rinsed in PBS, and again centrifuged at 300 x g for 10 min. Pellets were resuspended in 300 µL PBS-4% BSA and then incubated with a 1:100 dilution of fluorescein isothiocyanate-conjugated horse antimouse IgG for 30 min at 4 C on a shaker in the dark. After two additional wash/centrifuge cycles, the pellet was finally resuspended in 500 µL PBS containing 0.02% propidium iodide (Sigma Chemical Co., St. Louis, MO) for viability gating. Instrumentation used for flow cytometry consisted of a FACScan (Becton Dickinson, San Jose, CA) linked to a Consort 32 computer (Becton Dickinson).
Lysis II software (Becton Dickinson) was used for both data acquisition and analysis. Cells analyzed by flow cytometry were excited by an argon laser emitting 15 mW at 488 nm. Propidium iodide-stained cells were gated out during data collection. Green fluorescence of the remaining live cells was collected by a photomultiplier through a 530/30-nm bandpass filter. A logarithmic amplifier was used to compress the resulting fluorescence signal for storage and display on a four-decade log scale by Lysis II. Subsequent analysis of green fluorescence was used to determine the relative median fluorescence of each sample, calculated as follows. Cells incubated with fluorescent second antibody in the absence of primary antibody were used to determine median background fluorescence for each set of samples. This background fluorescence was then subtracted from the median fluorescence of each of the other samples in the set. The resulting adjusted median for each treatment condition was divided by the adjusted median of its control to obtain the relative median fluorescence of the stimulated sample as a multiple of the control. Statistical analysis included ANOVA with Scheffe’s correction of the mean of triplicate determinations.
Northern blot studies
Ishikawa cells exposed to E, E plus P, or increasing concentrations of EGF for 4–6 days were harvested and pelleted for Northern blot studies. Total ribonucleic acid (RNA) was extracted with TriReagent (Molecular Research Center, Cincinnati, OH). Equal amounts of RNA (20 µg lane) were separated by electrophoresis in 1.0% Maxi-Blot Agarose (Molecular Research Center, Cincinnati, OH) and transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH). The RNA was then cross-linked to the membrane by Stratalinker UV cross-linker 1800 (Stratagene, La Jolla, CA). Filters were incubated with prehybridization buffer \[50% formamide, 0.5% SDS, 1.0% glycine, 50 mmol/L NaPO4 (pH 6.5), 5 x Denhart’s solution, and 250 g/mL salmon sperm DNA\] for 2 h at 42 C and hybridized overnight at 42 C with 32P-labeled probe in hybridization buffer (50% formamide, 0.5% SDS, 10% dextran sulfate, 20 mmol/L NaPO4 (pH 6.5), 5 x SSC (standard saline citrate), 1 x Denhart’s solution, and 100 g/mL salmon sperm DNA). Filters were washed twice with 2 x SSC-0.1% SDS at room temperature for 5 min, then twice with 0.1 x SSC-0.1% SDS at 65 C for 30 min, and exposed to Kodak X-Omat film for 3 days at -70 C. A ß3-specific complementary DNA (cDNA) probe (provided by Dr. Clayton S. Buck, Wistar Institute, Philadelphia, PA) was prepared as follows: a 1.4-kb EcoRI/BamHI fragment of ß3 cDNA was [32P]deoxy-CTP labeled by the random priming method (Boehringer Mannheim, Indianapolis, IN). A specific cDNA probe for EGF receptor was provided by Dr. Shelton Earp (Chapel Hill, NC). Autoradiograms were performed to verify the removal of all prior radioactive probes before rehybridization with the glyceraldehyde-3-phosphate dehydrogenase cDNA probe to assess equal loading of RNA.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
vß3 integrin as shown
by immunocytochemistry in Fig. 1
. Fluorescence was not
present in the absence of primary antibody (Fig. 1A). The
vß3 integrin localized
to the pericellular borders and areas of cell-cell contact (Fig. 1B and
inset). A dramatic decrease in fluorescence intensity was
noted in cells treated with E and E plus P (Fig. 1C). E treatment alone
for up to 6 days was less inhibitory, and P alone had no effect (not
shown). To quantify relative expression of
vß3 under these
conditions, flow cytometry studies were undertaken. As shown in Fig. 2, combined E plus P treatment decreased relative
fluorescence intensity by 75% compared to a 45% decrease after
treatment with E alone.
|
|
vß3 in Ishikawa
cells, the effects of a variety of growth factors and cytokines were
examined using flow cytometry (Fig. 3). Whereas TGFß,
IGF-II, IL-1, IL-1ß, m-CSF, PDGF-AA, PDGF-BB, PDGF-AB, and TNF
had little or no effect on
vß3 expression, EGF,
TGF, and bFGF dramatically increased the
vß3 relative median
fluorescence (P < 0.01). These findings were confirmed
by immunofluorescence staining (Fig. 1D
, EGF); similar results were
obtained with TGF and bFGF. Subsequent experiments focused on the
effects of EGF on vß3
expression.
|
vß3 occurred at the
level of transcription, Northern blots were performed using E and/or P
and EGF (10 ng/mL). As shown in Fig. 4A, basal
expression of the ß3-subunit in Ishikawa cells
was diminished by both E and P, consistent with earlier results. In
response to EGF, transcription of the
ß3-subunit increased in a dose-dependent
manner. The predominant species was a 5.2-kilobase (kb) band, although
two smaller and lesser bands (3.0 and 1.7 kb) were also present and
appeared to be regulated. These latter species probably represent
alternatively spliced forms of the ß3-subunit.
As shown in Fig. 4B, Ishikawa cells also expressed the EGF receptor
(EGF-R) which was present at the expected 7.7-kb size, but the EGF-R
did not appear to be regulated by either sex steroids or EGF in this
cell line.
|
vß3, a
dose-response curve was produced by flow cytometry (Fig. 5). Stimulation of
vß3 expression was
dose dependent, with maximum stimulation occurring at concentrations of
1 ng/mL EGF or more (P < 0.01). To investigate the net
effects of steroid inhibition and EGF-induced up-regulation of
vß3, Ishikawa cells
were cultured in the presence of E and P with or without increasing
concentrations of EGF. Treatment with E and P decreased
vß3 expression
relative to levels observed in control cells (untreated), but EGF
treatment negated this effect in a dose-dependent manner that was
maximal at 1 ng/mL (Fig. 6).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
vß3 integrin, is
regulated directly or indirectly by steroid hormones and these shifts
in ER and PR (17). In addition, there is growing appreciation for the
roles of various growth factors in the implantation process (23, 24, 30, 31). Although the factors that regulate endometrial receptivity
remain somewhat obscure, studies employing recently identified markers
of receptivity, like certain members of the integrin family of cell
adhesion molecules, offer the means to investigate events surrounding
the periimplantation period (32).
The lack of a suitable in vitro model of human endometrium
has limited this area of study. Our previous work with the Ishikawa
cell line has validated its use as a model system for studies of
hormonally regulated events in endometrial epithelium (28, 29). These
well differentiated adenocarcinoma cells have both functional ER (26, 27) and PR (28). Northern blot studies have demonstrated that E exerts
transcriptional control over PR expression in a manner similar to that
seen in native endometrium. Ishikawa cells also express the same
complement of integrins as that in normal endometrium in
vivo. Both constitutive (2ß1,
3ß1, and
6ß4) and cycle-dependent
(1ß1 and
vß3) integrins (29)
are present on these cells. Moreover, the ability to inhibit and
stimulate the expression of these marker proteins in Ishikawa cells
makes them a useful tool for studies of the implantation process.
In the present study we have demonstrated that the
vß3 integrin is
dynamically regulated both by the sex steroids E and P and by growth
factors. Whereas the morphological development of the secretory
endometrium is clearly dependent on E and P, its functional maturation
[as reflected by vß3
expression (17) and embryo receptivity] temporally coincides with a
sharp decline in epithelial ER and PR (21, 22). These observations
suggest that the expression of
vß3 and perhaps other
critical proteins is inhibited by E and P and beginning only after
receptors for these steroids in the endometrium are effectively
down-regulated.
Growth factors and cytokines are important known regulators of
integrins. Endothelial cells, for example, exhibit increased expression
of 2ß1,
3ß1,
5ß1, 6ß1,
6ß4, and
vß5 in response to bFGF and
simultaneous down-regulation of 1ß1 and
vß3 (33). The
vß3 integrin, recently
demonstrated to play an important role in angiogenesis (34), is
stimulated by bFGF (35). In the present study, both bFGF and EGF
significantly increased expression of
vß3 in Ishikawa cells.
Although bFGF does not exhibit cycle-dependent changes (36), evidence
that EGF plays a role in mammalian implantation is accumulating. Data
derived from studies in mice localize EGF or EGF-like molecules to the
luminal epithelium before implantation and show that EGF mediates
estrogen-induced uterine cell proliferation (37, 38). The EGF-R is
present on the trophoblast during implantation in the rabbit (39), and
EGF or EGF-like molecules are expressed and enhance blastocyst
development in the mouse at the time of implantation (37, 40). Local
injection of EGF promotes nidation in the delayed implantation rat
model (41), and both EGF and EGF-R are present in human endometrium,
decidua, and placenta (42, 43).
In the present study, the role of EGF in stimulating the
vß3 integrin was
studied in detail. Using both flow cytometry and Northern blot
analyses, we noted a dose-dependent increase in expression of the
ß3-subunit. Of interest, the inhibitory
influence of E plus P treatment could be overcome by concomitant
treatment with EGF. Ongoing studies using the promoter for
ß3 in a chloramphenicol acyltransferase
reporter gene construct have confirmed that EGF is stimulatory and the
sex steroids are inhibitory to ß3-subunit
promoter activity (44).
In summary, we investigated the effects of sex steroids and growth
factors on expression of the
vß3 vitronectin
receptor in the Ishikawa endometrial cell line. Regulation of this
integrin, which normally appears at the time of implantation in human
endometrium, may involve both suppression by the sex steroids E and P
and stimulation by growth factors, including EGF. Our data suggest that
regulation occurs at the transcriptional level. Evidence suggests that
development of a receptive endometrium involves a cascade of events
orchestrated by hormones and growth factors, including up-regulation of
vß3 expression during
the periimplantation period. The Ishikawa cell line would appear to be
a useful model for studies of the mechanisms involved in human embryo
implantation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Division of Reproductive Endocrinology, Department
of Obstetrics and Gynecology, Suite 404 1245 Highland Avenue, Abington
Memorial Hospital, Abington, Pennsylvania 19001.
Received January 6, 1997.
Revised March 24, 1997.
Accepted March 28, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1ß1 integrin. J Steroid Biochem Mol Biol. 59:31–39.[CrossRef][Medline]
vß3 prevents maturation of
blood vessels during embryonic neovascularization. J Cell Sci. 108:2655–2661.[Abstract]
v integrins. Science. 270:1500–1502.This article has been cited by other articles:
 |
|
 |
|
  S. Bai, G. Grossman, L. Yuan, B. A. Lessey, F. S. French, S. L. Young, and E. M. Wilson Hormone control and expression of androgen receptor coregulator MAGE-11 in human endometrium during the window of receptivity to embryo implantation Mol. Hum. Reprod., February 1, 2008; 14(2): 107 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C K Wang, R S Robinson, A P F Flint, and G E Mann Quantitative analysis of changes in endometrial gland morphology during the bovine oestrous cycle and their association with progesterone levels Reproduction, August 1, 2007; 134(2): 365 - 371. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Achache and A. Revel Endometrial receptivity markers, the journey to successful embryo implantation Hum. Reprod. Update, November 1, 2006; 12(6): 731 - 746. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Mo, A. E. Vendrov, W. A. Palomino, B. R. DuPont, K.B.C. Apparao, and B. A. Lessey ECC-1 Cells: A Well-Differentiated Steroid-Responsive Endometrial Cell Line with Characteristics of Luminal Epithelium Biol Reprod, September 1, 2006; 75(3): 387 - 394. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. A. Lessey Endometrial Responsiveness to Steroid Hormones: A Moving Target Reproductive Sciences, February 1, 2004; 11(2): 61 - 62. [PDF]
|
|
|
|
 |
|
 C. W. Gregory, E. M. Wilson, K. B. C. Apparao, R. A. Lininger, W. R. Meyer, A. Kowalik, M. A. Fritz, and B. A. Lessey Steroid Receptor Coactivator Expression throughout the Menstrual Cycle in Normal and Abnormal Endometrium J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2960 - 2966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.B.C. Apparao, L. P. Lovely, Y. Gui, R. A. Lininger, and B. A. Lessey Elevated Endometrial Androgen Receptor Expression in Women with Polycystic Ovarian Syndrome Biol Reprod, February 1, 2002; 66(2): 297 - 304. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. T. Arnold, B. A. Lessey, M. Seppala, and D. G. Kaufman Effect of Normal Endometrial Stroma on Growth and Differentiation in Ishikawa Endometrial Adenocarcinoma Cells Cancer Res., January 1, 2002; 62(1): 79 - 88. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. B. C. Apparao, M. J. Murray, M. A. Fritz, W. R. Meyer, A. F. Chambers, P. R. Truong, and B. A. Lessey Osteopontin and Its Receptor {alpha}v{beta}3 Integrin Are Coexpressed in the Human Endometrium during the Menstrual Cycle But Regulated Differentially J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4991 - 5000. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Bowen and J. S. Hunt The Role of Integrins in Reproduction Experimental Biology and Medicine, April 1, 2000; 223(4): 331 - 343. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. L. Larimore and J. B. Stanford Postfertilization Effects of Oral Contraceptives and Their Relationship to Informed Consent Arch Fam Med, February 1, 2000; 9(2): 126 - 133. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kimmins and L. A. MacLaren Cyclic Modulation of Integrin Expression in Bovine Endometrium Biol Reprod, November 1, 1999; 61(5): 1267 - 1274. [Abstract] [Full Text]
|
|
|
|
 |
|
 R.R. Gonzalez, A. Palomino, A. Boric, M. Vega, and L. Devoto A quantitative evaluation of {alpha}1, {alpha}4, {alpha}V and {beta}3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal Hum. Reprod., October 1, 1999; 14(10): 2485 - 2492. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Leach, R. Khalifa, N. D. Ramirez, S. K. Das, J. Wang, S. K. Dey, R. Romero, and D. R. Armant Multiple Roles for Heparin-Binding Epidermal Growth Factor-Like Growth Factor Are Suggested by Its Cell-Specific Expression during the Human Endometrial Cycle and Early Placentation J. Clin. Endocrinol. Metab., September 1, 1999; 84(9): 3355 - 3363. [Abstract] [Full Text]
|
|
|
|
|
|
Y. Gui, J. Zhang, L. Yuan, and B. A. Lessey Regulation of HOXA-10 and its expression in normal and abnormal endometrium Mol. Hum. Reprod., September 1, 1999; 5(9): 866 - 873. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. T. C. Chen, S. Getsios, and C. D. MacCalman 17{beta}-Estradiol Potentiates the Stimulatory Effects of Progesterone on Cadherin-11 Expression in Cultured Human Endometrial Stromal Cells Endocrinology, August 1, 1998; 139(8): 3512 - 3519. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE |
