Mol. Hum. Reprod. Advance Access originally published online on June 4, 2008
Molecular Human Reproduction 2008 14(7):413-421; doi:10.1093/molehr/gan029
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Expression of immunomodulatory genes, their protein products and specific ligands/receptors during the window of implantation in the human endometrium
The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA, USA
1 Correspondence address. 601 Colley Avenue, Norfolk, VA 23507, USA. E-mail: oehninsc{at}evms.edu
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Abstract |
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We have demonstrated up-regulation of the immunomodulatory genes decay accelerating factor (DAF), interleukin 15 (IL-15) and osteopontin (OPN) during the window of implantation (WOI). Here, we characterized gene expression and determined the localization of their protein products and respective ligands at the opening and closure of the WOI. In addition, we used laser capture microdissection (LCM) to analyze the cell type-specific gene expression. Human endometrial biopsies from cycle Days 16, 21 and 24 were evaluated by real-time RT–PCR. Purified epithelial and stromal cells were obtained by LCM. Localization of the proteins and their ligands was assessed by immunohistochemistry. mRNA expression of DAF, IL-15 and OPN was significantly increased throughout the WOI. DAF, OPN and
vβ3 integrin were strongly immunolocalized to the glandular compartment by Days 21 and 24, whereas C3, IL-15 and IL-15R were highly stained in both glandular and stromal compartments. After LCM, gene expression of DAF was 4.8-fold increased in epithelium versus stroma, whereas for OPN there was a 2-fold increase. For IL-15, the expression in stroma was 8.7-fold higher than in epithelial cells. The progressive increase of the expression of these immunomodulatory genes, proteins and ligands during the WOI, support a critical role at the time of endometrial receptiveness. Key words: window of implantation/gene expression/immunomodulatory proteins/laser capture microdissection/human endometrium
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Introduction |
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The endometrium is receptive to embryonic apposition, attachment and invasion during a defined window that is temporally and spatially restricted, the so-called ‘window of implantation’ (WOI). Although there is still not full agreement as to the exact timing of embryo implantation in the human, clinical studies suggest that the window is temporally confined to Days 20–24 of a normal, ovulatory menstrual cycle (Navot and Bergh, 1991; Wilcox et al., 1999).
The endometrial lining must be prepared by estrogen priming and then differentiated by progesterone in a strict and carefully organized temporal sequence (Wilcox et al., 1999; Horcajadas et al., 2004a). The sex steroid hormones are responsible for the balanced production of a number of known and putative proteins such as growth factors, cytokines, and immunomodulators (Develioglu et al., 1999; Carson et al., 2000; Giudice, 2003). However, little is known about the specific and differential compartmental (stromal/epithelial) expression and localization of such critical factors in the human endometrium and their temporal variations throughout the WOI.
Previously, we characterized the gene expression profile of the pre-receptive and receptive whole endometrium during the normal natural cycle. We demonstrated up-regulation of 107 genes during the WOI, a number of which represented a group of immunomodulatory proteins, providing further support to the critical contribution of the maternal immune response to successful implantation (Mirkin et al., 2005; Zaret et al., 2006). Using similar gene array technology, other researchers have investigated gene expression of the receptive endometrium in the natural cycle comparing samples from the proliferative phase to the receptive phase (Kao et al., 2002; Borthwick et al., 2003; Riesewijk et al., 2003; Horcajadas et al., 2004b). Notwithstanding differences in study design, all these reports found several genes that were consistently up-regulated >2-fold in the receptive endometrium.
We elected to further study three genes and their protein products, including decay accelerating factor for complement (DAF or CD55), interleukin 15 (IL-15) and osteopontin (OPN or SPPI), highly up-regulated in the receptive versus the pre-receptive endometrium (Kao et al., 2002; Borthwick et al., 2003; Riesewijk et al., 2003; Horcajadas et al., 2004b; Mirkin et al., 2005), and their respective ligands (Complement 3 or C3, IL-15R and vβ3 integrin). The presence of estrogen and progesterone response elements in these genes suggests these steroid hormones may modulate their differential expression during the cyclical stages of endometrial receptiveness (Mirkin et al., 2005). The three selected genes/proteins have been reported to be involved in endometrial immunomodulation, with possible roles in implantation and fetal allograft tolerance (Zygmunt et al., 1998; Zourbas et al., 2001; Johnson et al., 2003; Al-Shami et al., 2005; Francis et al., 2006).
Tissues such as the human endometrium are comprised of multiple interacting cell populations whose components are difficult to study in isolation. In addition, different cell types within a complex target tissue such as the endometrium can respond dissimilarly when exposed to the same hormonal milieu. A new technology, laser capture microdissection (LCM), has expanded our ability to examine cell type-specific responses. LCM is a method for procuring a specific population of cells from a heterogeneous tissue section. As such, LCM is a powerful tool to approximate the true pattern of gene expression of the pure cell subpopulations in their actual tissue context.
Here, it was our objective to further characterize, temporally and spatially, gene expression and protein localization of DAF, IL-15 and OPN in the normal human endometrium during defined periods before (non-receptive endometrium) and at the time of opening and closure of the WOI (receptive endometrium, Days 21 and 24). The specific aims of these studies were: (i) to quantify the gene expression of these three genes; (ii) to immunolocalize their protein products and their known ligands in endometrial tissue sections and (iii) to characterize the gene expression profiles of isolated glandular and stromal cells following LCM.
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Materials and Methods |
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Human subjects
Endometrial biopsies were obtained from healthy volunteers participating as donors in our oocyte donation program. The subjects were regularly ovulatory women with normal menstrual cycles (eight parous and one nulligravida) and on no hormonal contraception. The mean age of the participants was 26.6 years (range 24–29). The Institutional Review Board of Eastern Virginia Medical School approved this study and written informed consent was obtained prior to recruitment and collection of biopsies. Biopsies were performed on the natural cycle based on detection of ovulation using a urinary LH surge detection kit (Assure LH Ovulation Predictor, Conception Technologies, San Diego, CA). Following previous studies, the day of the urinary LH surge was assigned LH = 0, and LH+1 was considered the day of ovulation or cycle Day 14 (Develioglu et al., 1999; Mirkin et al., 2004, 2005; Sherwin et al., 2007). At the time of the confirmation of the morning urinary LH surge in the clinic (LH = 0), the subject was randomized (using sealed envelopes) to schedule a single endometrial biopsy on cycle Day 16 (LH+3, pre-receptive endometrium, n = 3), 21 (LH+8, opening of the WOI, n = 3) or 24 (LH+11, closure of the WOI, n = 3). A total of nine volunteers were enrolled and a single biopsy was obtained on one or two passes of an endometrial suction pipelle (Unimar Pipelle, CooperSurgical, Shelton, CT) in the post-ovulatory period on days mentioned above. Two additional volunteers were biopsied during the late proliferative phase of the menstrual cycle for comparison purposes.
Endometrial samples
Each biopsy was divided into two samples: (i) immediately placed in OCT (Tissue-Tek, Sakura Finetek USA, Torrance, CA) and promptly frozen at –80°C in cryomolds (Tissue-Tek), and (ii) fixed in formalin for histological examination. An independent pathologist, blinded to the LH day, dated the biopsies using standard criteria (Noyes et al., 1950). The OCT-embedded tissue was cut into 8 µm sections using a cryostat (Tissue-Tek, model 4551A, Miles Inc, Elkhart, IN) at –30°C with a sterile blade. Sections were placed on frosted microscope slides (Fisher Finest Premium, Fisher Scientific, Pittsburgh, PA) and maintained at –80°C until further use.
RNA isolation and real-time RT–PCR
Total RNA was extracted from using an RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The quality of total RNA extracted was analyzed on an Agilent 2100 Bioanalyzer and quantification of total RNA was performed on a NanoDrop spectrophotometer.
cDNA was generated from 3 µl total RNA in a total volume of 20 µl containing: 2.5 µM random hexamers, 2.5 U/µl murine leukemia virus reverse transcriptase, 1 U/µl RNase inhibitor, 1x PCR buffer, 1 mM each deoxy-NTP and 5 mM Mg Cl2 (Applied Biosystems, Foster City, CA, USA). RT parameters were as follows: 23°C for 10 min, 42°C for 15 min (RT reaction), 99°C for 5 min (transcriptase deactivation), and 5°C for 5 min in an iCycler thermal cycler (BioRad, Hercules, CA). cDNA solutions were then stored at –20°C. Preparations without reverse transcriptase were used as negative controls, in which the absence of PCR products indicated a complete lack of contaminating genomic DNA.
Real-time RT–PCR (reverse transcription - polymerase chain reaction) was performed using a Lightcycler Fastart DNA Master Plus SYBR green I and a Lightcycler 2.0 instrument (Roche Applied Science, Indianapolis, IN) in a 20-µl total reaction volume, containing 2-µl cDNA and 0.5 µM of each sense and antisense primers (except 18S which was used at 0.3 µM). The primer source/sequence and the expected lengths of the resulting PCR products are shown in Table I. Primer utilized for OPN (or SPP1) and Cytokeratin (Cyk) were commercially available (Superarray Bioscience, Frederick, MD). All other primers were designed using Oligo Explorer or Primer 3 software and purchased from Invitrogen (Carlsbad, CA).
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Before amplification, samples were denatured at 95°C for 10 min. The template was amplified by 45 cycles of denaturation at 95°C for 10 s, annealing of primers at the specific temperature as given in Table I for 5 s, and extension at 72°C for 10 s, followed by a final extension at 72°C for 10 min. The melting protocol consisted of heating the samples to 95°C followed by cooling to 65°C for 15 s and slowly heating at 0.1°C per second to 95°C while monitoring fluorescence. Melting curve analysis was performed after each run to verify specific amplification. Product identity was confirmed by ethidium bromide-stained 3% agarose gel electrophoresis. Negative control consisted of PCR water replacing the cDNA solution (no template control). All PCR products exhibited a single peak in melting curves [Tm: 18S, 85.4; DAF, 84.3; IL-15, 80.7; OPN, 84.0; progesterone receptor (PR), 84.2; decorin (DCN), 86.0 and Cyk, 90.7, see Supplementary material, Fig. S1] and were identified as single bands of the appropriate size on ethidium bromide-stained agarose gel electrophoresis (Supplementary material, Fig. S2). In addition, specific amplification was confirmed by sequencing of PCR products purificated using QIAquick PCR purification kit (Qiagen). For each sample the amounts of the target gene and endogenous control gene (18SrRNA) were determined using a calibration curve. Standards (prepared using cDNA produced from total RNA extracted from pooled human endometrial biopsies) were defined to contain an arbitrary starting concentration and four serial dilutions 10-fold each were used to construct the standard curve. The amount of the target molecule was divided by the amount of 18SrRNA to obtain a normalized value. This ratio was reported as the relative mRNA expression. All determinations were performed in duplicate and standard curve dilutions were included in each run.
Immunohistochemistry
Immunohistochemistry was performed on 8-µm frozen sections of endometrial biopsies using MaxTag Histo Immunohistochemistry Kit (Rockland Immunochemicals, UK). We examined the immunolocalization of each protein and its ligand/receptor on cycle Days 16, 21 and 24 (DAF and its soluble ligand complement component 3 or C3, IL-15 and its receptor IL-15R, and OPN and its ligand vβ3 integrin). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min and the nonspecific binding sites were blocked with 2% normal goat serum (NGS) for 30 min at room temperature. Each primary antibody was serially diluted in a solution of PBS–2% NGS to optimize sensitivity and specificity. Tissue sections were incubated with primary antibody at 4°C overnight at the following dilutions: anti-DAF 8 µg/m (Abcam, Cambridge, MA, USA), anti-C3 1 µg/ml (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-IL-15 6 µg/ml (Abcam), anti anti-IL-15R 10 µg/ml (R&D Systems, Oxon, UK), anti-OPN 3.5 µg/ml (Novocastra Lab., Newcastle, UK) and anti-vβ3 integrin 6.5 µg/ml (Santa Cruz Biotechnology, Inc.,). After primary antibody incubation, sections were washed twice with PBS and incubated with biotinylated goat anti-mouse secondary antibody at a dilution of 1:120 for 30 min at room temperature. After rinsing with PBS, the immunoreactive antigen was visualized by incubating with avidin-biotinylated horseradish peroxidase (1:100) complex for 30 min and 3,3'-diaminobenzidine (0.5 mg/ml) as chromagen for 3 min to complete the reaction.
Negative controls included sections that were treated with a similar dilution of a non-immune mouse IgG1 (isotype control, eBioscience, San Diego, CA). Slides were counterstained with Mayer’s hematoxylin (Sigma, St Louis, MO, USA) followed by dehydration in a graded series of ethanol, cleared in xylene and mounted with mounting media. Immunohistochemical staining of the cells was evaluated semi-quantitatively in the same three biopsies for each cycle day. Intensity of immunostaining was scored as follows: no staining (–), low (1+), moderate (2+), strong (3+) and very strong (4+). Epithelial cells from the surface luminal layer and glands were considered together as endometrial epithelium. Representative fields were photographed at x200 and x400 magnification with an Olympus microscope (Olympus Corp., Tokyo, Japan) using an Olympus Q-color 3 camera. Two investigators (A.F. and J.Z.) scored the slides blindly and independently and results were averaged.
Isolation of purified populations of epithelial and stromal cells by LCM and analysis of gene expression
The expression of the three genes of interest was further examined in purified populations of glandular and stromal cells at the time of opening of the WOI (Day 21, n = 3 endometrial biopsies). Briefly, serial 8-µm sections of each endometrial biopsy were processed as follows: sections were fixed in 70% ethanol for 30 s, washed with RNase-free water, stained with Arcturus staining solution (Arcturus Engineering, Inc., Mountain View, CA) for 20 s, washed with RNase-free water, dehydrated in 75% ethanol 30 s, 95% ethanol 30 s, 100% ethanol 30 s and incubated 5 min in fresh xylene. Slides were wrapped air-dried for at least 20 min and immediately thereafter, an Arcturus PixCell II LCM system equipped with Olympus microscope (Olympus Corp.) was employed to capture glandular epithelial and stromal cells from the endometrial sections. A single LCM cap (CapSure HS LCM Caps, Arcturus) stored at room temperature was used per tissue section.
Optimal conditions for LCM capture of endometrial cells included a laser power of 60–80 mW, duration of 1.5–2.5 ms, and a laser spot size of 7.5 µm for glandular epithelium and 15 µm for stroma. Each LCM cap with captured cells was then tightly fitted to an Eppendorf tube containing lysis buffer (RNeasy, Qiagen), sited for 30 min in Arcturus block preheated at 46°C, then inverted several times and vortexed for 30 s. Lysates from various tissue sections from a single biopsy were pooled into a single Eppendorf tube, which was microcentrifuged after addition of the contents of each cap. At completion of the LCM process, samples were stored in lysate buffer overnight at –80°C, and RNA was extracted within 72 h. The entire cell capture process, from tissue sectioning to tissue lysis, was rapidly and sequentially completed to limit RNA degradation.
Total RNA was extracted from each sample using an RNeasy kit (Qiagen). The quality of total RNA extracted from epithelial and stromal cells isolated by LCM was analyzed on an Agilent 2100 Bioanalyzer. All samples (from whole tissue and after LCM) exhibited a RNA integrity number ranging from 6 to 7, reassuring quality for downstream applications (Fleige and Pfaffl, 2006). Quantification of total RNA after LCM was performed on a NanoDrop spectrophotometer, typical yields obtained ranged from 120 to 140 ng per dissection.
Contamination with non-target cellular components during LCM was monitored morphologically. In addition, to confirm the acquisition of a pure cell-type population (i.e. glandular epithelium and stroma), we quantified the mRNA corresponding to proteins strongly and specifically expressed by each cellular type. Cyk, a known marker of epithelial cells, was used as epithelial-specific marker. DCN was used as stromal-specific marker; according to previous publications DCN mRNA and protein are strongly expressed in stroma and absent from epithelium (Yanaihara et al., 2004).
Statistical analysis
Comparisons of gene expression levels of the secretory endometrium from pre-receptive endometrium (cycle Day 16) and receptive endometrium encompassing the WOI (Days 21 and 24), and epithelial versus stromal compartments after LCM, were performed using the Wilcoxon (nonparametric) test. Differences were considered statistically significant when P 
0.05. Data are presented as mean ± SEM.
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Results |
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Changes in gene expression of DAF, IL-15 and OPN in the endometrium throughout the WOI
The relative levels of DAF mRNA were 0.005 ± 0.002 on the proliferative phase, 0.015 ± 0.004 (mean ± SEM) on day 16, 0.255 ± 0.041 on day 21 and 0.692 ± 0.282 on day 24 of the menstrual cycle (Fig. 1A). For IL-15, the relative transcript levels were 0.013 ± 0.003 on the proliferative phase, 0.238 ± 0.040 on day 16, 0.688 ± 0.113 on day 21 and 0.830 ± 0.063 on day 24 (Fig. 1B). The OPN mRNA levels were 0.006 ± 0.003 on the proliferative phase, 0.043 ± 0.020 on day 16, 0.390 ± 0.236 on day 21 and 0.853 ± 0.095 on day 24 (Fig. 1C).
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Gene expression levels for all three genes during secretory phase cycle Days 21 and 24 were significantly higher than on cycle Day 16 (P < 0.03). Although there was a clear trend for all three genes toward increased mRNA levels on Day 24 compared with Day 21, these differences did not attain significance. Relative mRNA abundance, expressed as a ratio of mRNA levels on Day 21 and 24 over Day 16, showed increased expression throughout the WOI for all genes: 17- and 46-fold change for DAF, 3.1- and 5.3-fold change for IL-15, and 9.1- and 19.9-fold change for OPN, respectively, for Day 21 and Day 24.
Immunolocalization of DAF, IL-15 and OPN proteins and their specific ligands/receptors in the endometrium throughout the WOI
DAF and C3
Almost no DAF staining was found in the proliferative phase and a weak reaction was seen on cycle Day 16, with marked increase on Day 21 that was very strong and sustained on Day 24 (Fig. 2A–F). DAF protein was localized predominantly to the glandular and luminal epithelia with only weak staining of stromal cells. Staining of the endometrial epithelium was strongest in the luminal compartment, predominantly in the apical cellular regions.
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Immunohistochemical localization of C3 is shown in Fig. 2G–L. In the proliferative phase, the staining was weak-moderate and exclusively localized to the glands. Stromal patches expressing C3 were observed in the secretory phase (cycle Days 16, 21 and 24), where the prominent expression in both endometrial compartments (glands and stroma) was seen, with a very strong staining by Days 16 and 21. Glandular and luminal epithelial cells showed expression of C3 localized mostly to the apical region of the cells.
IL-15 and IL-15r
IL-15 and its specific receptor, IL-15R, were present in all days of the cycle in the secretory phase. For IL-15 (Fig. 3A–F), the differences between days were more pronounced in the epithelial cells (glands and surface) than in the stroma. In the proliferative phase very weak staining was observed, while on cycle Day 16, the epithelial cells showed a lower intensity compared with the stromal cells. The immunostaining intensity in the epithelial compartment (glands and surface) was higher on Days 21 and 24, and more prominent than in stroma.
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IL-15R
(Fig. 3G–L) was localized in the nuclear region as previously reported in other cellular types (Dubois et al., 1999). In the proliferative phase, weak immunostaining was observed in the epithelial cells, whereas stromal cells showed minimal patched immunoreactivity. Luminal epithelium showed increased staining on Day 16, and both stromal and glandular cells showed stronger staining by Day 21. A decreased expression was seen by Day 24 of the cycle, with patched immunoreaction in the stroma.
OPN and vβ3 integrin
For OPN, minimal immunoreactivity was observed in the proliferative phase, as well as in Day 16 of the menstrual cycle. OPN was strongly identified in the glands and surface epithelium by Days 21 and 24, with minimal staining of stromal cells. During the WOI stronger staining was observed on the apical region of the surface and glandular epithelial cells (Fig. 4A–F).
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Nearly identical temporal and spatial patterns were found for the OPN ligand,
vβ3 integrin, with almost no immunostaining observed in the proliferative phase. Its expression was markedly increased by Day 21, predominantly in the luminal and glandular epithelial cells, which were sustained by Day 24 (Fig. 4G–L). Immunostaining intensity scores are summarized in Table II.
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Gene expression of purified populations of epithelial and stromal cells obtained by LCM from receptive endometrium at opening of WOI
Since the expression of the three genes of interest was highest during the mid-secretory phase, endometrial biopsies from Day 21 were used for LCM. Each biopsy was carefully dissected by LCM for epithelial and stromal areas (Fig. 5). It was critical to confirm the purity of the isolated cell-type populations, and for that purpose we quantified the mRNA corresponding to proteins strongly and specifically expressed by each cellular type. The Cyk mRNA expression in epithelial cells was 8.1-fold higher than in stromal cells, whereas DCN expression was 9.2-fold higher in stromal versus epithelial cells similar to previously published reports. These data confirmed the purity of each cell population (Fig. 6).
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After confirming cell-type purity, mRNA expression was analyzed for DAF, IL-15 and OPN (Fig. 7). DAF relative expression was 4.8-fold higher in glandular than in stromal cells; OPN expression was also increased 2-fold when comparing glands and stroma. Conversely, IL-15 the expression in stroma was 8.7-fold higher than in glandular cells. In addition, PR mRNA expression was 3.2-fold higher in stromal than in epithelial cells.
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Discussion |
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We hypothesized that changes in immune response leading to successful embryo implantation are triggered by stimulation of gene expression at the crucial time of the opening of the window of receptivity. Two phenomena might occur thereafter: (i) gene ‘switch-on’ followed by ‘switch-off’, or (ii) ‘switch-on’ followed by continued stimulation of gene expression levels. Our results indicated that mRNA levels increased for DAF, Il-15 and OPN at the opening of the WOI (Day 21) and continued to increase at the time of theoretical closure of the window (Day 24). Since we detected a progressive increase in gene expression as well as in their protein products and their specific ligands/receptors, we propose that these proteins start their function at the time of putative attachment and continue to function in support of further implantation.
Decay accelerating factor
DAF is a glycophosphatidylinositol-anchored protein that binds to C3 and dissociates C3/C5 convertases, limiting C3a/C5a anaphylatoxin production and preventing progression of the complement cascade by C3b/C5b (Heeger et al., 2005). Since DAF is a complement-regulatory protein, two possible functions have been postulated: one, to mediate embryo/fetus protection from maternal complement-mediated attack; and second, to prevent epithelium destruction from increased complement expression by the time of implantation.
We observed a parallel increase of DAF mRNA and protein in the endometrium. Up-regulation of DAF during the WOI was demonstrated in several studies using cDNA microarrays, with expression increasing 5.9–34.5-fold (Kao et al., 2002; Borthwick et al., 2003; Riesewijk et al., 2003; Mirkin et al., 2005; Talbi et al., 2006) and also using semi-quantitative PCR (Lobo et al., 2004). Similar results were found by Young et al. (2002), who reported that immunohistochemical evaluation of DAF was minimal in the proliferative and early secretory phases and increased thereafter showing maximal expression in both glands and lumen by LH+8, persisting into menses. Our LCM data demonstrated that although the gene expression of DAF occurred in both compartments, the expression was 4.8-fold higher in the epithelium than in the stroma.
During the late proliferative phase, C3 was weakly expressed on the epithelial cells. This expression was markedly increased during the secretory phase, where the stronger immunostaining was seen by Day 16 and 21 in both stromal and epithelial compartments, similar to reports in the baboon (Sherwin et al., 2007). Previous reports showed a minimal expression of C3 in proliferative endometrium, since a prominent expression was observed in the secretory samples using in situ hybridization analysis (Sayegh et al., 1996) and immunohistochemistry (Hasty et al., 1994). Since C3 expression is increased by the receptive endometrium, the increased expression of DAF at the mid-luteal phase may function to protect the cells from increased C3 levels, preventing destruction of host cells.
Few clinical studies have related deficiencies of DAF and pathology. Patients with luteal phase defects were shown to have diminished DAF expression, which normalized after treatment with progesterone (Kaul et al., 1995). Comparing recurrent pregnancy loss patients with and without the diagnosis of antiphospholipid syndrome, a significant decrease in DAF both by quantitative PCR and immunohistochemistry in endometrial biopsies obtained on cycle Day 22–26 from women with antiphospholipid antibodies was reported (Francis et al., 2006). In addition, recent publications reported that C3 activation is required for antiphospholipid antibody-induced fetal loss, and is also involved in dysregulation of angiogenic factors (Holers et al., 2002; Girardi et al., 2006), identifying new effectors of immune-triggered pregnancy complications.
Interleukin 15
During early pregnancy, the main leukocyte population in the uterine mucosa (decidua) is CD56(+) CD16(–) natural killer (uNK) cells (Bulmer et al., 1991; King et al., 1991). In contrast to the circulating NK cells, which respond poorly to IL-15, this cytokine induces the proliferation of uNK cells, but no cytotoxicity against trophoblast (Verma et al., 2000). The broad expression of IL-15 compared with the expression of IL-2, suggests that IL-15 has activities beyond the immune system. In fact, it was reported that the invasion of cytotrophoblastic (JEG-3) cells is up-regulated by IL-15 in vitro (Zygmunt et al., 1998).
Here, we demonstrated a progressive increase in IL-15 mRNA expression from the proliferative through cycle Day 24, with significantly higher expression during the WOI. Observations of up-regulation of IL-15 and IL-15R in secretory, compared with proliferative endometrium, have been reported previously (Lobo et al., 2004). IL-15 protein has been reported to be present in both the stroma and the glands (Kitaya et al., 2000) and is therefore a prime candidate for stimulating uNK cell proliferation in vivo. These cells provide the main mechanism by which the maternal immune system recognizes the trophoblasts cells (Moffett and Loke, 2006).
Our gene expression data in LCM samples appear not to correlate with the immunostaining intensity, suggesting that the main source of IL-15 secretion corresponds to some of the cellular type components within the stromal compartment, and that after secretion, the protein can bind to the epithelial cells. This postulate is in agreement with the findings of the IL-15 receptor (IL-15R) localization to both cell compartments.
To the best of our knowledge, this is the first report on the immunohistochemical localization of IL-15R in the human endometrium. IL-15R showed nuclear localization. This protein was previously reported to be present in isolated uNK cells by RT–PCR and Southern blot analysis (Verma et al., 2000); here, we report immunolocalization of IL-15R to epithelial and stromal cells.
Eight differentially spliced human IL-15R variants have been identified (Dubois et al., 1999), with the full-length IL-15R associated primarily with the nuclear membrane, and part of the receptor having an intranuclear localization. Isoforms lacking exon 2, which encodes a protein-binding sushi domain with a putative nuclear localization signal, showed extranuclear localization and were unable to bind IL-15 (Dubois et al., 1999). Here, we have localized IL-15R to the nucleus; we speculate that we are detecting isoforms that contain the sushi domain, maintaining the capability of the receptor to bind IL-15, therefore participating in the regulation of IL-15 action.
In a recent publication, Roberts et al. (2005) reported similar mRNA and protein levels in stromal and epithelial cells. The existence of different IL-15 isoforms and primers used for PCR quantification may be some of the reasons for an apparent disagreement between the above cited report and our results. Because the synthesis and secretion of IL-15 is controlled at multiple levels in addition to transcription (Tagaya et al., 1997; Gaggero et al., 1999), the mRNA levels may not necessary reflect the levels of protein secretion. In addition, it has been suggested that a complex between the non-secretory IL-15 isoform and IL-15R may regulate not only IL-15, but also IL-15R gene expression (Nishimura et al., 2005).
Osteopontin
OPN is a secreted glycosylated phosphoprotein member of the family of extracellular matrix proteins. OPN has been identified in endometrial glands, immune cells and uterine secretions during the secretory phase. Microarray studies consistently identified up-regulation of OPN 4.9–20-fold (Carson et al., 2002; Kao et al., 2002; Borthwick et al., 2003; Riesewijk et al., 2003; Mirkin et al., 2005; Talbi et al., 2006) during the mid-secretory phase. In a well-differentiated human endometrial adenocarcinoma cell line known to possess receptors for estrogen and progesterone (Ishikawa cells), OPN expression was increased by progesterone and progesterone plus estradiol treatment (Apparao et al., 2001).
vβ3 integrin has been recognized as the primary receptor for OPN, resulting in cell-to-cell attachment and cell spreading via cytoskeletal changes (Miyauchi et al., 1991). The specificity of binding between OPN and vβ3 integrin was demonstrated by Apparao et al. (2001) using Ishikawa cells. Adhesion of the Ishikawa cells to OPN was reduced only in the presence of blocking antibodies to vβ3 integrin.
In our study, OPN mRNA levels showed low level on cycle Day 16, and a markedly increase on Days 21 and 24. Clearly, increased protein expression was observed in the luminal and glandular epithelium throughout the WOI. Nearly identical temporal and spatial patterns were found for vβ3 integrin. The mRNA expression after LCM showed 2-fold higher levels in glandular epithelium versus stroma. OPN up-regulation by the mid-luteal phase takes place at the same time the PR is down-regulated in the epithelium (Lessey et al., 1988), suggesting that progesterone may have an indirect regulative effect via stromal paracrine factors, as has been previously proposed (Koji et al., 1994; Bruner et al., 1995; Young et al., 2002). This is in accordance with our finding that PR expression in cycle Day 21 biopsies was 3.2 times higher in stroma than in epithelium. In addition, it has been recently reported that a major localization of OPN is within a subpopulation of granulated uNK cells during progression of decidualization (Herington and Bany, 2007). A functional link is supported by the dramatic decrease in OPN expression in decidua of uNK cell-deficient IL-15–/– compared with normal IL-15+/+ mice (Ashkar et al., 2003).
In conclusion, our results indicated increased gene expression from the pre-receptive through the receptive endometrium. Because of the relatively small number of samples analyzed, a statistical significant change was not found between cycle Day 21 and 24 mRNA levels, but there was a clear trend for the three immunomodulatory genes to be up-regulated at the onset of the WOI with a progressive increase throughout the putative closure of the WOI. Furthermore, their specific proteins products, as well as their ligands/receptors, demonstrated similar temporal variations suggesting that these proteins have a function at the crucial time of endometrial receptiveness. The immunolocalization of these proteins and their receptors to the compartments of the endometrium and the cell-type specific gene expression pattern within the different compartments sets the stage for future studies to assess endocrine and autocrine/paracrine modulation. It is envisioned that as the technologies of genomics and proteomics advance, studies of gene expression and protein identification and localization within the specific endometrial compartments will yield improved knowledge of endometrial proteins, and their post-transcriptional modifications, which are essential for the establishment of the WOI in the human (Oehninger, 2008).
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Supplementary data |
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Supplementary material is available at molhr Journal online.
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Funding |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary dataFundingReferences |
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This work was partly supported by Organon, Inc., USA.
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Submitted on March 10, 2008; resubmitted on May 7, 2008; accepted on May 9, 2008.
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