Hum. Reprod. Advance Access originally published online on October 24, 2007
Human Reproduction 2007 22(12):3223-3231; doi:10.1093/humrep/dem216
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Progesterone withdrawal up-regulates fibronectin and integrins during menstruation and repair in the rhesus macaque endometrium
1 Northwestern University, Chicago, IL 60611, USA 2 Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health and Science University, 505 NW 185th Avenue, Beaverton, OR 97006, USA
3 Correspondence address. Tel: +503-690-5331; Fax: +503-690-5563; E-mail: brennerR{at}ohsu.edu
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Abstract |
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BACKGROUND: Fibronectin (FN) is a component of the extracellular matrix that participates in wound healing in various tissues as an adhesive ligand for integrins (Itgs). To determine whether these molecules play similar roles during menstrual repair, we evaluated the expression and localization of FN and specific Itgs in the primate endometrium under hormonally controlled conditions.
METHODS: Ovariectomized rhesus macaques were treated for 2 weeks with estradiol (E2) followed by E2 with progesterone for 2 weeks. On day 28, progesterone was withdrawn and uteri were collected during menstruation, postmenstrual repair, and the proliferative and secretory phases. Analysis was by focused microarray, real time PCR, in situ hybridization and immunocytochemistry.
RESULTS: Progesterone withdrawal induced significant elevations of FN, Itg 
5 and Itg 
1 transcripts during menstruation as compared to day 28 (FN: P < 0.01; Itg 5: P < 0.05; Itg 1: P < 0.05; real time PCR). These increases were concentrated in the glandular epithelium (FN) and stroma (Itg 51) of the uppermost zones. Cyclic changes in Itg 3 occurred in the glandular epithelium.
CONCLUSIONS: Spatially and temporally restricted peaks of expression of FN and its Itg receptors are closely correllated with menstruation and postmenstrual repair in the primate endometrium.
Key words:
fibronectin/integrin /integrin /endometrium/menstruation
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Introduction |
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Progesterone withdrawal at the end of the menstrual cycle up-regulates various inflammatory cytokines, prostaglandins, vascular endothelial growth factor and several matrix metalloproteinases (MMPs). The latter are considered the proximate cause of menstrual breakdown, and there is an extensive literature on these and other destructive changes induced by progesterone withdrawal in the endometrium (Marbaix et al., 1996
; Salamonsen, 2003; Jabbour et al., 2006). Much less is known about the reconstructive processes involved in menstrual repair, which consists of re-epithelialization, surface healing and cessation of bleeding.
The endometrium that remains after menstruation has a ragged and torn surface marked by multiple gland openings surrounded by denuded stromal elements that lack a covering epithelium (Ludwig and Spornitz, 1991). Healing of this denuded stromal surface begins with the transformation of the uppermost gland cells into a migratory phenotype. Cells move out from the necks of the glands, spread out and meet migrating cells from other glands to form a new luminal surface (Ludwig and Spornitz, 1991). This process is complete by 5 days after progesterone withdrawal at which time estrogen-dependent mitotic activity begins in the uppermost regions of the glands, which initiates the next proliferative phase (McClellan et al., 1990).
The underlying cellular and molecular mechanisms that support this form of ‘wound healing’ are not well understood. A recent review of the literature on menstrual repair concluded that ‘...almost nothing is known about how endometrial repair is achieved after menstruation or parturition’ (Salamonsen, 2003).
In other wounded tissues, particularly skin (O'Toole, 2001), a variety of molecules, including fibronectin (FN), collagens, laminin and vitronectin are involved in the healing process (Clark, 1996). FN is a large fibrillar glycoprotein secreted usually as a homodimer composed of two 
250 kDa monomers linked together by a pair of disulfide bonds. Interactions between FN and specific integrins (Itg) are known to enhance cell adhesion and migration during wound healing (Kim et al., 1992; Gehlsen and Argraves, 1988; Livant et al., 2000). FN, which exists both in a soluble form in plasma (Mosher, 1984) and a fibrillar form in the extracellular matrix (ECM), contains various domains (types I, II and III) that harbor specific binding sites for different Itgs (Hohenester and Engel, 2002). These binding sites include arginine-glycine-aspartic acid (RGD) and the proline-histidine-serine-arginine-asparagine (synergy site or PHSRN) sequences, located in the type III domain of the FN polypeptide chain.
Itgs, large heterodimeric glycoproteins comprised of and units, form a family in mammals that comprises 24 distinct Itgs that provide cells with anchorage, traction for migration, and signals for polarity, position, differentiation and possibly growth (Ruoslahti and Pierschbacher, 1987). The 1 subunit can bind to a number of different units to form heterodimers such as 5 1 (Beliard et al., 1997; Miyata et al., 2000; Bischof et al., 2006), 3 1 (Chintala et al., 1996; O'Toole, 2001) and some others that can all function as FN receptors.
A recent review of cell migration (Schwartz and Horwitz, 2006) notes that FN-Itg-1 interactions induce the GTPases Rac and Cdc 42, which interact with intracellular actin to regulate a coordinated series of cell membrane protrusions and adhesions which propel a cell across a substratum. Moreover, the concentration of FN on the substratum has dramatic effects on migration speed. In many cell types, migration speed is highest at intermediate concentrations of FN and lowest at both low and high FN concentrations (Lauffenburger and Horwitz, 1996). Novel cellular models to explain these effects have been developed (Gupton and Waterman-Storer, 2006). The signaling pathways between FN, Itgs and intracellular protein components of focal adhesions including talin, vinculin and actin have recently been reviewed (Critchley et al., 1999; Wozniak et al., 2004).
Many reports indicate that FN and various Itgs are expressed by the endometrium (Grinnell et al., 1982; Lessey et al., 1992; Mularoni et al., 1992; Beliard et al., 1997; Reddy and Mangale, 2003) but we found no studies that focused specifically on the expression and cellular localization of FN and FN receptors during menstruation and repair in the non-human primate model. In a first effort to gain insight into these processes, we analysed samples of rhesus macaque endometrium with pathway-focused arrays of human genes associated with the ECM and found that FN and Itg 1 transcripts were dramatically increased during menstruation and repair compared with the late secretory phase of the cycle. Subsequently we used real time PCR, in situ hybridization (ISH) and immunocytochemistry to extend these findings. Here, we report that FN and specific FN receptors are spatio-temporally regulated in the uppermost endometrial zones during menstruation and repair in the rhesus macaque endometrium.
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Materials and Methods |
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Experimental animals
Animal care and husbandry was provided by the veterinary staff of the Division of Animal Resources of the Oregon National Primate Research Center (ONPRC), Oregon Health and Science University (OHSU). All studies and procedures were reviewed and approved by the ONPRC/OHSU Institutional Animal Care and Use Committee. Artificial menstrual cycles were induced in 42 ovariectomized macaques by implanting the animals sequentially with Silastic capsules containing estradiol (E2) for 14 days, and then E2+progesterone for 14 days as described previously (Nayak and Brenner, 2002
). In this model, removal of the progesterone implant at the end of the artificial cycle induces menstruation and begins the next cycle. The implants used in these experiments produced serum levels of 80.7 ± 43 pg/ml (mean±SEM) E2 and 4.9 ± 1.1 ng/ml progesterone, which are within the normal range for cycling rhesus macaques. The entire uterus was collected at the following times: menstruation, menstrual repair/early proliferative phase, late proliferative phase, early secretory phase and late secretory phase. Other ovariectomized macaques were treated with E2 and progesterone as above, but both the E2 and progesterone implants were removed at the end of the cycle. These animals are referred to as Hormone Deprived (HD) and they undergo typical menstruation and repair in the absence of E2 as well as progesterone. In the HD group, uteri were sampled on days 2 and 3 after both E2 and progesterone were removed. The overall sampling schedule including the number of animals per group is shown in Table 1.
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Tissue collection
Immediately after necropsy, the uterine corpus was cut in half along the longitudinal axis from fundus to cervix with a single edged razor blade. Each uterine half was then quartered by another longitudinal cut along the same axis. Cross-sectional slices (1.0–2.0 mm thick) of the endometrium were made perpendicular to the longitudinal axis of each quarter to provide a full thickness sample of the endometrium from lumen to myometrium. Some of the cross sections were microwave irradiated for 7 s, embedded in Tissue Tek II Optimal Cutting Temperature (OCT) compound (Miles Inc. Elkhart, IN), and frozen in liquid propane for immunocytochemistry, as previously described (Nayak et al., 2005
). Some were also fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.3) and embedded in paraffin. For ISH, similar samples were embedded in OCT and frozen in liquid propane without microwave irradiation. The remaining endometrium from each animal was separated from the myometrium with fine scissors, frozen and stored over liquid nitrogen for subsequent RNA assay.
Isolation of total RNA
Samples were thawed in 10 volumes of TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and immediately homogenized with a Polytron tissue homogeniser (Brinkmann Instruments, Westbury, NY, USA) and extracted with the standard TRIzol protocol for total RNA. The TRIzol-extracted sample was precipitated with ethanol, combined with RNeasy lysis buffer (Qiagen, Valencia, CA, USA) and purified with the RNeasy mini kit. RNA bound to the RNeasy filters was treated with RNase-free DNase (Catalog no. 79 254; Qiagen) on the filter following the manufacturer's instructions. Concentrations of total RNA in the final extract were quantified by ultraviolet absorbance on a 640B spectrophotometer (Beckman Instruments Inc., Fullerton CA, USA), and RNA integrity was determined on an Agilent 2100 Bioanalyzer (Aligent Technologies; Palo Alto, CA, USA).
Pathway-focused gene arrays
Total endometrial RNA was analysed on GEArray Q Series Human extracellular matrix and adhesion molecule arrays (HS 101; SuperArray, Bethesda, MD, USA). The arrays contain 96 target complementary DNAs (cDNAs) associated with cell adhesion and regulation of the ECM, with three sets of constituently expressed genes (cyclophilin, RPL-13a and -actin) and negative control DNA (PUC 18) to detect non-specific hybridization. Total RNA (2 ug) was reverse transcribed and amplified using the GEArray AmpoLabeling-LPR Kit (Catalog number L-03; SuperArray) for radioactive detection. The cDNA was radiolabeled with [32P] 2'-deoxycytidine 5'-triphosphate (dCTP) (Perkin Elmer, Shelton, CT, USA), denatured at 94°C and hybridized to the arrays in a roller hybridization incubator (model 1000, Robbins Scientific, Sunnyvale, CA, USA) overnight at 60°C in 3.5 ml hybridization buffer. After high stringency washing was as follows: (0.1x SSC, 0.5% SDS @ 60°C for 15 min x12) to remove unhybridized probe, the arrays were visualized by phosphor imaging with a Bio-Rad Molecular Imager FX (Bio-Rad Laboratories, Hercules, CA, USA) and quantified with ScanAlyze (Michail Eisen, Stanford University, Stanford, CA, USA). For each array, the mean intensity for the negative controls (PUC 18 and no cDNA) was calculated, and this signal level with 2 SD of the mean intensity was considered background level on the blot. The background was subtracted from signal for each target cDNA and the relative signal for each gene was calculated as the ratio of intensity of target gene to the intensity of RPL-13a (signal ratio).
Real-time quantitative RT–PCR
The first strand cDNA was synthesized using a hexamer random primer according to the instructions in the Promega, USA kit. Real-time quantitative PCR was performed using the relative standard curve method on an ABI 7900HT using Taqman Master Mix (G01 418, Applied Biosystems). The reaction was performed in 10 µl with 0.1 µM cDNA (or about 2% of the product of RT), 0.25 µm gene specific primers for FN, Itg 5 and Itg 1 (Morrison et al., 1948) (Table 2). To control for the amount of total RNA in each reaction and to normalize the target signal, 18S mRNA was used as an active endogenous control (Young et al., 2002). Gene specific Taqman probes (0.3 µM) were labeled at the 5' ends with either fluorescent FAM for targeting genes or fluorescent VIC for the house keeping genes, and TAMRA (quencher) at the 3' ends (Table 2). The results were expressed in relative terms as the ratio of target gene:18S RNA. Each sample was run in triplicate. The PCR conditions were 50°C 2 min, 95°C 10 min, then 40 cycles of 95°C 15 min and 60°C 1 min.
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in situ hybridization
ISH of frozen sections of macaque endometrium was conducted with macaque-specific riboprobes labeled with [35S] UTP (PerkinElmer Life and Analytical Science) for FN and the Itgs. Briefly, the probes were prepared from PCR-amplified, monkey-specific transcription templates in two steps. Initially PCR products were amplified using primers shown in Table 2. A T7 RNA polymerase promoter was ligated to gene-specific double-stranded cDNA of PCR product using the Lig-N-Scribe no-cloning promoter addition kit (Ambion, Austin, TX, USA). In some cases the T7 RNA polymerase promoter was added to the specific primers at the time of design. The specific sense and antisense templates were amplified by PCR for 30 cycles. Free primers and salts were removed from the templates with Qiagen's PCR spin columns. The templates were sequenced by the ONPRC Molecular Biology Core on a Genetic Analyzer 3100 to verify the accuracy and size of the PCR products. Finally, the riboprobes were synthesized with the templates by in vitro transcription using a MaxScript kit (Ambion). The macaque-specific cDNA and conceptual protein sequences were submitted to The National Center for Biotechnology Information Genbank database [Core Nucleotide Access # AY833433 [GenBank] (FN); AY901982 [GenBank] (
5); AY878076
[GenBank]
(1)].
Techniques for ISH with [35S]UTP-labeled probes were as described previously (Slayden et al., 2004). Briefly, frozen sections, 5 µm thick, were mounted on Super Frost Plus slides (Fisher Scientific, Pittsburgh, PA, USA) and fixed in 4% paraformaldehyde in PBS for 10 min at 4°C. The tissue sections were rinsed in 2x saline sodium citrate (SSC), acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min and then air dried. One slide per tissue group was treated with RNase A [20 mg/ml, 0.5 M NaCl, 0.01 M Tris, 1 mM EDTA (pH 8.0)] as a negative control. All the slides were prehybridized for 1 h at 42°C in 10 mM dithiothreitol, 0.3 M NaCl, 20 mM Tris (pH 8.0), 5 mM EDTA, 1x Denhardt solution, 10% dextran sulfate and 50% formamide. Sections were then incubated at 55°C overnight in the same solution containing the appropriate concentration of the antisense probe (5 x 106 cpm/ml). Representative slides were also incubated with sense probe as another negative control. After hybridization, all the slides were treated with RNase A at 37°C for 30 min to inactivate non-hybridized probe, rinsed in a descending series of SSC (2x, 1x and 0.5x SSC) and then washed in 0.1x SSC at 65°C (high stringency) for 30 min. Sections were dehydrated in an ascending series of alcohol dilutions, vacuum dried, coated with NTB2 autoradiographic emulsion (Eastman Kodak), stored at 4°C for 2 weeks, developed in aqueous D-19 (Eastman Kodak), lightly counterstained with hematoxylin, dehydrated in an ascending series of alcohol dilutions, cleared with xylene and coverslipped with Permount (Fisher Scientific).
Immunocytochemistry
Immunocytochemistry for FN, Itg5, Itg3 and Itg1 was performed in cryosections with general procedures previously described in detail (Brenner et al., 2003). Briefly, fresh tissues were microwaved for 7 s before being embedded in OCT, frozen in liquid propane and cryosectioned at 5 µm. Cryosections were mounted on SuperFrost Plus slides (Fisher Scientific) and fixed in 2% paraformaldehyde in phosphate buffer (pH 7.3) for 10 min at room temperature. To inhibit endogenous peroxidase activity, the slides were incubated with a solution containing glucose oxidase (1 U/ml), sodium azide (1 mM/l) and glucose (10 mM/l) in PBS for 45 min. The slides were then incubated with blocking serum for 20 min and with an endogenous biotin blocking solution (Vector Laboratories Inc., CA, USA) for 30 min. The slides were then incubated with the first antibody, either mouse anti-FN monoclonal antibody at 20 µg/ml (clone FBN11, Labvision Co., CA, USA) mouse anti-human Itg 51 monoclonal antibody at 0.1 µg/ml (clone HA5, Chemicon, CA, USA), mouse monoclonal anti-Itg 5 at 5 µg/mL (clone P1D6, Chemicon), mouse monoclonal anti-Itg 3 at 1 µg/ml (clone P1B5, Calbiochem, CA, USA) or mouse monoclonal anti-Itg 1 at 0.1 ug/ml (clone DE9, Chemicon) all at 4°C overnight. After rinsing and immersion in blocking serum again, sections were incubated with a biotinylated horse anti-mouse antibody (Vector Laboratories Inc.) for 30 min at room temperature. The slides were then rinsed in PBS and incubated in ABC solution (Vector Laboratories Inc.) for 60 min and then washed in Tris-HCl (pH 7.6). The slides were incubated in 0.025% 3,3'-diaminobenzidine/4 HCl (Wako Chemicals, Richmond, VA, USA) in Tris-HCl buffer containing 0.03% hydrogen peroxide (Fisher Scientific) and 0.026% osmium tetroxide for 10–15 min and rinsed again in Tris buffer followed by deionized water. After rinsing in 38 mM Tris-HCl (pH 7.6) and deionized water, the sections were postfixed with 2% paraformaldehyde and lightly counterstained with Meyer's hematoxylin to facilitate identification of cellular elements.
In our laboratory, all antibodies are tested at a series of dilutions on various tissues fixed in various ways, and include positive and negative controls as recommended by the manufacturers and otherwise as selected by us. For example, Labvision recommends kidney as a positive control for their FN antibody, which we used. In addition, the endometrial literature made clear that FN and its receptors were present in human endometrial stroma, so we also confirmed that these antibodies worked on human endometrium as an additional positive control.
Imaging technique
Low-power micrographs were photographed with an Olympus OM-system 38 mm macro lens (Olympus Optical, Tokyo, Japan) on Ektachrome 64-T film (Eastman Kodak) and digitized with an Epson Expression 10 000 XL scanner (Epson Corp., Long Beach CA, USA). High-power micrographs were captured through Zeiss planapochromatic lenses with a Leica DFC 480 camera (Leica, Wetzlar, Germany) Digital images were adjusted for sharpness and contrast with Adobe Photoshop (Adobe Systems, Seattle, WA, USA), and photomicrographs were printed with a Stylus Photo 1280 printer (Epson, Tokyo, Japan).
Statistical analysis
Real-time quantitative PCR data were presented as relative expression of target gene:18S mRNA (mean ± SEM). One-way analysis of variance followed by the Tukey test was performed for multiple comparisons using GraphPad Software, Prism 3.02 (GraphPad Software, San Diego, CA, USA).
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Results |
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Pathway-focused gene array analysis
Of the 96 genes on the array, 33 transcripts were significantly increased
4-fold or more over background during the menstrual, as compared to the late secretory, phase. Most of these elevated transcripts were MMPs and tissue inhibitors of metalloproteinases known to increase during menstruation in the macaque (Rudolph-Owen et al., 1998). Of the remaining transcripts, FN and Itg1 showed the largest increases in expression. For example, FN levels, expressed as the signal ratio of FN to RPL13A mRNA, increased from 0.05 to 2.54 during the transition from the secretory to the menstrual phase, a 50-fold increase, and the signal ratio for Itg1 increased from 0.13 to 2.17, a 17-fold increase.
Real-time PCR analysis
Real-time PCR analysis confirmed that FN gene expression (Fig. 1) was significantly elevated during days 2–3 after progesterone withdrawal compared to levels in the late secretory phase (P < 0.01). After menstruation, FN levels declined gradually to a minimum during the late secretory phase. In the HD group there was a similar significant peak in FN expression on days 2–3 after withdrawal of both progesterone and E2 (P < 0.05), indicating that the increase in FN expression after progesterone withdrawal did not require E2. Real-time quantitative PCR also revealed a significant elevation of both Itg 1 (Fig. 2) and Itg 5 (Fig. 3) gene expression during 2–3 days after progesterone withdrawal compared to levels in the secretory phase. Itg 1 gradually declined from its expression peak and was significantly reduced by the end of the proliferative phase (P < 0.05; Fig. 2). Itg 5 declined in parallel from its peak to a minimum during the proliferative phase (P < 0.01; Fig. 3). In the HD group, there were similar peaks of expression of both Itg 5 and Itg1 after withdrawal of both progesterone and E2 indicating that these increases did not require E2.
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Cellular localization of FN
In situ hybridization
During the late secretory phase (day 28), FN mRNA was only weakly expressed by the glands and stroma, though strongly expressed by the spiral arteries (Fig. 4). By 2–3 days after progesterone withdrawal, as menstrual breakdown occurred, there was a marked increase in the glandular FN mRNA signal that was restricted to the uppermost endometrial glands and luminal epithelium. The stromal FN mRNA signal was weak until around day 5, when it increased in the uppermost stroma while the luminal epithelial signal remained strong. Subsequently, the luminal and glandular epithelial FN mRNA signal declined and remained at very low levels through the rest of the cycle. The stromal cells continued to express FN mRNA through the rest of the cycle but their expression was never as strong as several days after progesterone withdrawal. FN mRNA expression in the spiral arteries was strong but showed minimal change throughout the cycle. In the HD group, the expression pattern of FN mRNA during menses and repair was essentially identical to that seen when E2 was present after progesterone withdrawal (not shown).
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Immunocytochemistry
On day 28 of the secretory phase, staining for FN protein was strong in the stroma and only weakly expressed by the glands (Fig. 5). By 2–3 days after progesterone withdrawal, staining of the uppermost glands had increased considerably and consisted of positively stained granules, consistent with the FN mRNA signal shown by ISH. Around day 5, FN protein staining was still evident in the luminal epithelium and the uppermost stroma but the uppermost glands had become negative, and they remained negative through day 14. For the rest of the cycle, FN protein staining was minimal in the luminal epithelium and glands, but remained strong in the stroma, particularly as a component of the basal lamina surrounding the glands. The endothelial cells in the spiral arteries also strongly expressed FN protein (not shown) but showed little change throughout the cycle. In the HD group, FN protein staining followed a similar pattern, consistent with the results of ISH (not shown).
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Cellular localization of Itgs
In situ hybridization
Itg
1Fig. 6 shows that on day 28, Itg
1 expression was very low in the stroma while moderate in the glands, and that progesterone withdrawal induced a very strong up-regulation of Itg 1 mRNA expression on days 2–3 that was greatest in the stroma of the uppermost zones. Stromal expression stayed high in the uppermost zone through days 5–6 and then declined to low levels by day 14. Glandular and luminal epithelial cells expressed Itg 1 throughout the remainder of the cycle at moderate levels. Immunocytochemical staining of Itg 1 showed similar patterns (not shown). Itg 1 was also expressed by vascular endothelial cells without major change throughout the cycle. Fig. 6 also shows that in the HD group, the expression pattern of Itg 1 on day 2 was essentially identical to that seen when E2 was present after progesterone withdrawal. Higher magnification images (Fig. 7) confirm that after progesterone withdrawal the greatest increase in Itg 1 expression was in the stroma, not the epithelium, and that stromal expression became low by the eighth day of the cycle and remained so through the rest of the cycle.
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Immunocytochemistry
Itg
51Two different mouse monoclonal antibodies against this FN receptor were used. One clone (colone P106, Chemicon) was made against the separate form of Itg
5 and the other clone (51, Chemicon) was prepared against heterodimeric Itg 51. The latter antibody gave the most consistent results and showed that this FN receptor was primarily expressed by the stromal cells. Itg 51 stromal staining was strongest in the uppermost zones throughout the cycle and was especially strongly expressed on days 3 (Fig. 8A) and 5–6 (Fig. 8B) after progesterone withdrawal. Also, the endothelial cells of the spiral arteries expressed Itg 51 quite strongly but this signal showed little change during the cycle.
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Itg
3Because Itg
51 was primarily expressed by the stroma, not the glandular or luminal epithelium, we used immunocytochemistry to examine the expression of Itg 3, another FN receptor (O'Toole, 2001). In the case of Itg 3, stromal cells were negative and the luminal and glandular epithelial cells were strongly positive. For example, on day 28 of the cycle the glands were stained evenly with the Itg 3 antibody throughout the thickness of the endometrium, from the uppermost functionalis to the deep basalis zones (Fig. 9A). After progesterone withdrawal on days 1–3, Itg 3 staining remained strong in the deeper zones but was greatly reduced in the sloughing, menstruating zones (Fig. 9B). By day 5, as the surface healed, staining for Itg 3 was renewed in the luminal and glandular epithelium (Fig. 9C) and remained strong through day 8 (Fig. 9D) and the rest of the cycle. As typical for an Itg, the Itg 3 signal was localized within epithelial cell membranes (Fig. 10). In the HD group (both progesterone and E2 withdrawn), Itg 3 staining was lost from the uppermost endometrial zones around day 2 exactly as when only progesterone was withdrawn (not shown).
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Discussion |
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An adhesion hypothesis
Our working hypothesis was that the endometrium, in response to progesterone withdrawal and the subsequent tissue destruction, would synthesize molecules crucial to the repair process, that these molecules would be concentrated at the endometrial surface where healing occurs, and that this process would not require estrogen. The latter follows because the endometrium heals in the absence of estrogen when menstruation is induced by ovariectomy.
Our results showed that progesterone withdrawal did indeed induce a significant rise in FN, a major adhesive molecule, that this increase occurred initially in the upper portions of the glands, followed later in the uppermost stroma, and that estrogen was not required for these increases. The secretion of FN during and after menstruation would therefore provide abundant quantities of an adhesive molecule within the upper regions of the ragged endometrium. In addition, there were parallel increases in Itg 5 and Itg 1 expression that occurred predominantly in the uppermost zones. Consequently, an important FN receptor, Itg 51 (Hohenester and Engel, 2002), was in the right place at the right time to modulate cell movements.
However, antibodies specific to Itg 5 primarily marked stromal cells, not epithelial cells, while ISH of Itg 1 showed expression in both stromal and epithelial cells. Because Itg 31 can also function as a FN receptor (O'Toole, 2001), we examined the distribution of Itg 3 and found that this Itg was strongly expressed throughout the cycle only in the surface and glandular epithelium, not in the stroma. However, during menstrual breakdown, the Itg 3 signal was greatly reduced in the uppermost, fragmenting zones, while remaining strong in the deeper glands. Presumably, the various lytic processes associated with menses led to a loss of Itg 3 protein from the epithelial cell membranes, which would likely favor gland breakup during sloughing. During surface repair, around day 5, the Itg 3 signal returned to the luminal and glandular epithelium and remained strong throughout the rest of the cycle. We concluded that Itg 31 could serve as an epithelial receptor for FN while Itg 51 could serve as a stromal FN receptor. As noted, the temporal and spatial increases in FN and Itgs that occurred after progesterone withdrawal were essentially similar whether E2 was maintained or withdrawn. Estrogen is essential for the glandular mitosis that begins in the upper zones on the fifth day after progesterone withdrawal (McClellan et al., 1990) but not for menstruation or surface healing.
This is the first report that the endometrial glands synthesize FN shortly after progesterone withdrawal, that both FN and Itg 51 expression peak around the time of menses and repair, that their expression occurs in a marked gradient, strongest in the uppermost and weakest in the lowest endometrial zones and that these events are similar in the presence and absence of estrogen. It is also the first observation in the rhesus macaque that Itg 31 is expressed cyclically by the luminal and glandular epithelium of the endometrium. FN could facilitate the migratory movements of both epithelial and stromal cells through interactions with Itg 31 and Itg 51, respectively. Full validation of this hypothesis awaits experimental blocking studies with either FN antibodies (Kaspar et al., 2006) 1 Itg antibodies (Bergelson et al., 1992) or RGD peptides known to antagonize FN and Itg-dependent adhesive interactions (Enomoto-Iwamoto et al., 1997; Ochsenhirt et al., 2006).
A corollary hypothesis—FN and MMPs
When progesterone is withdrawn at the end of the cycle there are large increases in expression of MMPs in the uppermost endometrial zones including MMP-1, 2 and 3, considered to be key enzymes synthesized by stromal cells around days 2–4 (Rudolph-Owen et al., 1998). These enzymes, among others, are presumed to play key roles in the destruction of the ECM during menses and to participate in glandular remodeling (Malemud, 2006).
FN is known to induce MMP-2 expression through an Itg-mediated, signal transduction pathway (Hoffmann et al., 2006). Consequently, immediately after progesterone withdrawal, the increase in FN may enhance MMP-2 expression and thereby facilitate breakdown of the ECM during menses. Conversely, FN is specifically targeted by MMP-3 for proteolysis (Fukai et al., 1995) and proteolytic fragments of FN, though weak as adhesive ligands, have unique biological effects, including induction of apoptosis in various tissues (Fukai et al., 1995). Consequently, FN may act during menstruation to induce MMP-2, and may also be a source of biologically active fragments that facilitate apoptosis, all in addition to its potential role as an adhesive molecule later on during repair. The strong up-regulation and secretion of FN by the glands that occurs with the onset of menstruation is consistent with this corollary hypothesis.
In summary, progesterone withdrawal sets in motion a dramatic up-regulation of FN and its Itg receptors in the uppermost endometrial zones. These molecules may not only mediate the migratory and adhesive processes that accompany postmenstrual surface healing and repair but may also play roles in menstrual breakdown itself. Other components of the ECM and other Itgs, including 4, 3 and 5, which can also serve as FN receptors, may also play important roles in menstruation and menstrual repair. Additional research on these and other factors and on the molecular mechanisms involved in these processes is needed to expand our knowledge of how the endometrium repeatedly heals, repairs and regenerates itself.
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Acknowledgements |
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The authors thank, Andrea Lawson for help with photomicrography and Yibing Jia of Molecular Core Laboratory for his technical support. Supported by NIH HD 43 209, HD 18 185 and RR-00 163.
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References |
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Submitted on April 5, 2007; resubmitted on June 11, 2007; accepted on June 18, 2007.
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