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Endocrinology Vol. 148, No. 2 618-626
Copyright © 2007 by The Endocrine Society

Identification of Novel Genes Regulated by Chorionic Gonadotropin in Baboon Endometrium during the Window of Implantation

J. R. A. Sherwin, A. M. Sharkey, P. Cameo, P. M. Mavrogianis, R. D. Catalano, S. Edassery and A. T. Fazleabas

Department of Obstetrics and Gynaecology (J.R.A.S.), The Rosie Hospital, Cambridge CB2 2SW, United Kingdom; Department of Obstetrics and Gynecology (P.C., P.M.M., A.T.F.), University of Illinois at Chicago, Chicago, Illinois 60212-7313; Department of Pathology (A.M.S., R.D.C.), Cambridge CB2 1QP, United Kingdom; and Proteomics Core Laboratory (S.E.), Rush University Medical Center, Chicago 60612

Address all correspondence and requests for reprints to: Asgi T. Fazleabas, Ph.D., HCLD, Professor of Physiology, Director, Center for Women’s Health & Reproduction, Department of Obstetrics & Gynecology, University of Illinois, 820 South Wood Street, Chicago, Illinois 60612-7313. E-mail: asgi{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chorionic gonadotropin (CG) is an early embryo-derived signal that is known to support the corpus luteum. An in vivo baboon model was used to study the direct actions of human CG (hCG) on the endometrium, during the periimplantation period. Endometrial gene expression was analyzed using microarrays. The endometrial biopsies were taken from hCG-treated (n = 5) and control (n = 6) animals on d 10 after ovulation. Class comparison identified 61 genes whose transcript levels differed between control and hCG-treated samples (48 increased, 13 decreased in mean expression level more than 2.5-fold; P < 0.01). Real-time PCR of transcript abundance confirmed up-regulation of several of these, including SerpinA3, matrix metalloproteinase 7, leukemia inhibitory factor (LIF), IL-6, and Complement 3 (P ≤ 0.05). Analysis of protein abundance in endometrial flushings showed increased LIF and IL-6 protein in uterine flushings from hCG-treated animals compared with controls. Complement C3 and Superoxide dismutase 2 that were also up-regulated, were further evaluated by immunocytochemistry. Complement C3 showed a marked increase in stromal staining in response to hCG, whereas and superoxide dismutase 2 localization was most markedly increased in the glandular epithelial cells. Expression of Soluble Frizzled Related Protein 4, the most highly down-regulated gene, was also validated by PCR. Our experiments have shown that hCG induces alterations in the endometrial expression of genes that regulate embryo attachment, extracellular matrix remodeling and the modulation of the immune response around the implanting blastocyst. Several of these genes, including LIF and gp130, have been shown to be essential for implantation in other species. This study provides strong evidence that the preimplantation embryo itself influences the development of the receptive endometrium via secreted paracrine signals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHORIONIC GONADOTROPIN (CG) plays a pivotal role in embryo implantation, by supporting corpus luteum function (1, 2). Human CG (hCG) is synthesized by the preimplantation embryo and previous studies using a baboon model, have demonstrated the ability of hCG to induce several of the morphological changes that are seen in the endometrium during early pregnancy, such as epithelial plaque formation (3). These changes can be inhibited by a progesterone receptor antagonist (4) and require synergy between ovarian and endometrial responses to hCG because epithelial plaque formation is not seen in ovariectomized animals (3). However, the direct action of hCG on endometrium is responsible for the induction of stromal cell differentiation, stromal edema, and also the up-regulation of {alpha}-smooth muscle actin, a marker of decidualization (3, 5). Recent studies have also shown that hCG acts in synergy with IL-1ß, an endometrially expressed, proinflammatory cytokine, to induce IGF binding protein 1 (IGFBP-1), another marker of decidual differentiation (6). The human uterus also contains LH/hCG receptors in multiple cells types with the highest levels in epithelial cells during the secretory phase (7, 8). In the baboon, epithelial expression of LH/CGR is also highest during the mid-secretory phase and cell-specific changes in receptor distribution are further regulated by hCG, as well as decidualization (9). The activation of hCG receptor in human endometrial cells in vitro results in up-regulation of COX-2, increased stromal cell differentiation, modulation of pro- and antiimplantation cytokine production, together with an increase in blood flow through vasodilatation and angiogenesis (8).

Given the importance of hCG production in reproductive success (10), we hypothesized that the direct action of hCG on primate endometrium, might also induce gene expression changes that would enhance endometrial receptivity and support embryo implantation. Here we report changes in endometrial gene expression and protein synthesis after hCG infusion in a baboon model. Some of the genes that are shown to be regulated by hCG are already known to play important roles in the various stages of embryo implantation. However, we have also identified many new genes that were not previously known to be regulated in the endometrium by hCG. These genes may play a role in tissue remodeling, immune modulation, and antioxidant defense systems and represent new biological functions that are regulated in the endometrium by paracrine signals from the implanting primate embryo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant hCG was obtained from Dr. A. F. Parlow (National Hormone and Pituitary Program, Harbor-UCLA Medical Center, Torrance, CA) and from Serono Laboratories (Rockland, MA). Mouse monoclonal antibodies against Complement C3 (C3) and superoxide dismutase 2 (SOD2) for immunohistochemistry were obtained from Gene Tex, Inc. (San Antonio, TX). The ELISA kits for measuring leukemia inhibitory factor (LIF) and IL-6 were purchased from Biosource International (Camarillo, CA).

Tissue collection
All experimental procedures were approved by the Animal Care Committee of the University of Illinois (Chicago, IL). Uterine tissue was obtained from six control and five hCG-treated adult female baboons (Papio anubis), by endometriectomy or hysterectomy, on d 10 post ovulation (PO). Ovulation was detected in cycling female baboons by measuring peripheral serum levels of estradiol, beginning 7 d after the first day of menses. The day of the estradiol surge was designated as d –1, with d 0 as the day of the ovulatory LH surge and d 1 as the day of ovulation. On d 5 PO, an oviductal cannula was attached to an Alzet osmotic minipump and recombinant hCG was infused at the rate 1.25 IU/h for 5 d, as previously described (3). The experiment was terminated on d 10 PO (which corresponds to the expected day of implantation in pregnant baboons). At the time of surgery, uterine flushings were obtained by perfusing the uterine lumen with 5 ml of sterile Ca2+- and Mg2+-free Hanks’ buffered salt solution, as previously described (6). For the ELISA uterine flushings were obtained from five control animals and four hCG-treated baboons. Two of the five controls and two of the four hCG-treated animals, are from the same animals used in the array analysis.

Histology and immunohistochemistry
Tissues were fixed in 10% buffered formalin and subsequently embedded in paraffin. Sections were cut at 5 mm and mounted onto SuperFrost Plus microscope slides (Fisher Scientific, Hanover Park, IL). Sections were dried on a slide warmer overnight. Sections were deparaffinized in 2 changes of xylene, rehydrated in graded ethanol concentrations and rinsed in TBS. Immunostaining was done using the Vectastain Elite Kits (Vector Laboratories, Burlingame, CA). Briefly, sections were blocked in 3% normal serum and incubated in primary antiserum overnight at 4 C after antigen retrieval in citrate buffer (pH 6.0). The antibody for C3 was used at a concentration of 10 µg/ml and the SOD antibody at 52 µg/ml. Sections were then incubated for 30 min at room temperature with appropriate biotinylated secondary antibodies (1:200). Sections were washed and incubated with avidin biotin complex for 30 min at room temperature. Staining was visualized by immersing sections in 3,3'-diaminobenzidine tetrahydrochloride solution containing 0.03% H2O2. Nuclei were counterstained with Gill’s hematoxylin and the slides were dehydrated and coverslipped. Slides were analyzed and photographed using a Nikon microscope and Spot Digital camera and software (Nikon, Melville, NY).

RNA extraction from endometrial samples
Total RNA was extracted from each endometrial tissue using Trizol Reagent (Invitrogen Life Technologies, Carlsbad) according to the manufacturers’ instructions. RNA quality was assessed by loading 200 ng of total RNA onto an RNA Labchip and analyzed on an A2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany).

Preparation of fluorescence-labeled targets and cDNA microarray hybridization
One microgram of total RNA from each sample was used to synthesize double-strand cDNA (ads-cDNA) using the SMART PCR cDNA synthesis Kit (Clutch). The ads-cDNA from 3 control animals was labeled with Cy3-deoxyuridine triphosphate, using a Bioprime DNA labeling kit (Life Technologies) with random hexamer primers, based on the protocol by Petalidis et al. (11). This pooled mixture of cDNAs acted as a common reference for each of the 11 array experiments. Each of the control and hCG-treated endometrial cDNAs was labeled separately with Cy5-deoxyuridine triphosphate (Amersham, Little Chalfont, UK). The Cy3 and Cy5-labeled ads-cDNA were individually purified using Autoseq G50 columns (Amersham) and then each Cy5-labeled probe and an aliquot of the Cy3-labeled common reference was pooled with 10 µg/ml human Cot-1 DNA (Life Technologies), 1 µg/ml Poly dA (Amersham), before hybridization to a cDNA microarray at 50 C for 16 h. The human cDNA microarrays used (HMN2) consist of 8000 cDNAs and were produced in the Department of Pathology, University of Cambridge. Sequence-verified human cDNA inserts were PCR-amplified from bacterial lysates using vector-specific primers and the purified PCR products were printed onto GAPSII aminosilane slides (Corning) in 150 mM phosphate pH 8.5/0.01% sodium dodecyl sulfate (SDS) buffer using a BioRobotics 610 MicroGrid II robot and MicroSpot 2500 quill pins producing a spot size of approximately 140 µm in diameter. Slides were fixed after printing by baking on a hot plate at 80 C for 2 h. They were then blocked by immersion in 1% BSA (molecular biology grade, B 2518; Sigma, St. Louis, MO) + 0.1% SDS in 3x standard saline citrate (SSC) for 20 min at 65°, and then denatured by immersion in water at 95° for 2 min. Slides were finally immersed in isopropanol before drying by centrifugation. After hybridization the arrays were washed twice in 2x SSC, 0.5% SDS and twice in 0.1x SSC, 0.1% SDS for 20 min each at room temperature. The fluorescence signal on microarrays was acquired using a Genepix 4100 microarray scanner (Axon Instruments, Foster City, CA). The scanned images were processed using GenePix Pro 3.0 software (Axon Instruments).

Array analysis
Microarray data analyses were performed using Biometric Research Branch Array Tools developed by Dr. Richard Simon and Amy Peng (National Cancer Institute, Biometric Research Branch, Division of Cancer Treatment and Diagnosis) (http://linus.nci.nih.gov/BRB-ArrayTools.html). Intensity values from the duplicate spots are averaged and an intensity filter was used to remove very low signal spots (if both channels resulted in a signal >50). Lowess intensity-dependent normalization was used to adjust for differences in labeling intensities of the Cy3 and Cy5 dyes. The adjusting factor varied over intensity levels (12, 13).

Genes that were differentially expressed among the two classes were identified using a random-variance t test. The random-variance t test is an improvement over the standard separate t test because it permits sharing information among genes about within-class variation without assuming that all genes have the same variance (14). Genes were considered statistically significant if their P value was less than 0.01. A stringent significance threshold was used to limit the number of false-positive findings. This univariate T test resulted in 191 genes that are significant at P < 0.01. The genes were further selected form the t test result by using a mean expression level cutoff of than 2.5, which identified 61 genes whose transcript levels differed between control and hCG-treated samples (48 increased, 13 decreased)

Real-time PCR verification
To verify the results obtained from the cDNA microarray, real-time PCR (Taqman) verification was performed for seven genes: SerpinA3, matrix metalloproteinase 7 (MMP7), Leukemia Inhibitory Factor (LIF), gp130 IL-6, C3, and Soluble Frizzled Related Protein 4. Details of primers and probes are given in Table 1Go. For SerpinA3, C3, and SFRP4, real-time PCR was performed with probes labeled with 5'FAM and 3'TAMRA using 2x PCR mix from Abgene according to manufacturer’s instructions (Abgene; Epsom, UK). Other cDNAs were quantified using SYBRgreen PCR mix from Abgene. Standard curves were generated by serial dilution of a standard preparation of total RNA produced from pooled samples of cDNA from baboon endometrium. cDNA was produced from each control or hCG-treated endometrial sample by RT using 3 µg of total RNA with 200 IU Superscript RT (Invitrogen Life Technologies). The expression values obtained were normalized against those from the control ribosomal 18S to account for differing amounts of starting material. Expression levels in the hCG and control-treated tissues were compared using the Mann-Whitney (nonparametric) test. Differences were considered statistically significant when P < 0.05.


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  		TABLE 1. Primers and probes used for Taqman

 
Detection of LIF and IL-6 in uterine flushings
The concentration of LIF and IL-6 in uterine flushings was measured using ELISA kits (human IL-6 and LIF/HILDA ELISA from Biosource International) according to the manufacturer’s protocol. The minimum detectable dose for hIL-6 is less than 2 pg/ml, and the assay does not cross-react with human IL-1{alpha}, IL-1ß, IL-2, IL-3, IL-4, IL-7, IL-8, IL-10, G-CSF, GM-CSF, IFN-{alpha}, IFN-{gamma}, LIF, MIP-1ß, MCP-1, OSM, RANTES, TGF-ß, and TNF{alpha}. The IL-6 ELISA intraassay coefficient of variation was 5.1–7.7% and the interassay coefficient of variation was 6.5–9.3%. The minimum detectable dose for LIF is 20 pg/ml and the assay does not cross-react with human IL-1{alpha}, IL-1ß, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, G-CSF, GM-CSF, IFN-{alpha}, LIF, MIP-1ß, MCP-1, OSM, TGF-ß, TNF{alpha}, and TNFß. The LIF ELISA intraassay coefficient of variation was 4.5–5% and the interassay coefficient of variation was 6–8.4%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of genes up-regulated in endometrium after treatment with hCG
Endometrial cDNAs from hCG-treated (n = 5) and control animals (n = 6) were hybridized to HMN2, cDNA arrays and the detected intensity signals were normalized and statistically analyzed using the Biometric Research Branch Array tools. Data above background signal were obtained for 4800 of the 8000 of the arrayed cDNAs. Class comparison was performed using a two-sample t test with random variance that showed that the data from one of the control cDNA arrays were discordant with the other control data. cDNA from this control animal had not been used to generate the common pooled reference and the data from this control array were not used in subsequent analysis. Class comparison identified 61 genes whose transcript levels differed between the remaining control (n = 5) and hCG-treated (n = 5) samples by more than 2.5-fold (P < 0.01). Forty-eight transcripts were increased and 13 decreased (see Fig. 1Go and Table 2GoGo).


Figure 1
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  		FIG. 1. The scatter plot shows the distribution of normalized mean signal intensity for the Cy3 and Cy5 signal for each spotted cDNA from HMN2 microarrays hybridized with fluorescently labeled cDNA probes synthesized from baboon endometrium. The endometrial test samples had been treated in vivo with control vehicle (A) or CG (B). These test samples were labeled with Cy5 (y-axis) and each sample was hybridized to a separate array at the same time as a common reference RNA labeled with Cy3 (x-axis). The hybridization signal for each spot was determined for both Cy3 and Cy5 and mean values are plotted after normalization. The majority of genes are equally expressed in the test and the common reference samples and give a Cy5/Cy3 ratio of one. The green lines on either side of this denote a mean change in signal intensity of 2-fold in the test samples compared with the common reference. Selected genes whose expression differs in the control and CG-treated samples are arrowed.

 

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  		TABLE 2A. Genes up-regulated following in vivo CG treatment of baboon endometrium

 

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  		TABLE 2B. Genes down-regulated after in vivo CG treatment of baboon endometrium

 
To validate changes in abundance of some of these RNA transcripts, real-time RT-PCR was performed for SerpinA3, MPP-7, Leukemia Inhibitory Factor (LIF), gp130, Superoxide dismutase-2 (SOD2), Soluble Frizzled Related Protein-4 (SFRP4), C3, and also IL-6. IL-6 is part of the LIF family of cytokines and is also represented on the HMN2 microarray. However, it was not shown to be differentially expressed between the control and hCG-treated samples, despite the hybridized signal intensity being above background in all arrays. Real-time PCR analysis using the same RNA samples as used for the array analysis confirmed a statistically significant increase in gene expression in the hCG-treated endometrial cDNA abundance, for SerpinA3, MMP-7, LIF IL-6 and C3 (Mann-Whitney test; P ≤ 0.05; see Table 3Go). SFRP-4 expression was shown to decrease (4.9-fold decrease; P = 0.05). Although transcript levels for gp130 and SOD2 showed an increase after hCG treatment by real-time RT-PCR analysis, this did not reach significance with these small groups (Mann-Whitney test, P = 0.13 and 0.08 respectively; Table 3Go).


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  		TABLE 3. Real-time quantification of selected transcript abundance in baboon endometrium, treated in vivo with CG or control.

 
Quantitation of LIF and IL-6 protein abundance in uterine flushings
ELISA was used to quantify LIF and IL-6 protein abundance in uterine flushings obtained just before the endometrial biopsy on d 10 PO (Control; n = 5 and hCG treated; n = 4). IL-6 protein concentration was significantly increased after hCG administration when compared with controls at the same time point (median 172 pg/ml (range 45–333) vs. 3pg/ml in control animals (range 1–57); Mann-Whitney test P = 0.019; see Fig. 2AGo). The amount of LIF protein in the uterine flushings on d 10 PO, also showed a tendency to increase in the hCG-treated animals; however, this was not statistically significant (318 pg/ml in hCG-treated animals (range 60–789), compared with median 81 pg/ml (range 35–113) in control animals (Fig. 2BGo).


Figure 2
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  		FIG. 2. Quantification of IL-6 and LIF abundance in baboon uterine flushings in control animals and after in vivo treatment with CG. Mean cytokine abundance (±SEM) in baboon uterine flushings taken from animals treated in vivo with control and sampled on d 10 PO (D10 control) or CG and sampled on d 10 (D10 + CG). The increase in IL-6 protein abundance on d 10 after hCG is significant, P = 0.02. LIF protein abundance on d 10 appeared to increase after CG treatment but this was not significant due to the small number of animals.

 
Immunolocalization of C3 and SOD
In addition to PCR analysis, C3 and SOD were immunolocalized in endometrial tissues from hCG-treated and control animals. Immunocytochemical analysis of C3 demonstrated a marked increase in stromal staining with a marked decrease in epithelial staining compared with untreated controls (Fig. 3Go, A–D). In contrast, SOD staining was markedly increased in the glandular epithelium in response to hCG (Fig. 3Go, E and F).


Figure 3
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  		FIG. 3. Immunolocalization of C3 and SOD in the uterine endometrium from control baboons at d 10 PO and those treated with hCG. C3 immuostaining is present in both epithelial and stromal cells in control animals (A and C), whereas in response to CG the stromal staining is most intense (B and D). SOD staining was present in the glands during the normal cycle (E) but increased markedly in the glands and stroma in response to hCG stimulation (F).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Embryo-derived signals act to rescue the corpus luteum and also to orchestrate endometrial adaptations to embryo implantation (1). However, the specific effects of hCG on the endometrium are poorly understood. Blastocyst attachment to the apical surface of the luminal epithelium is a multistep process mediated in a sequential manner by chemokines and adhesion molecules (15). CG is known to act on endometrium via its receptor to regulate some genes involved in the development of a receptive endometrium for embryo implantation and decidualization (3, 9, 16). However, in this study we have shown that it also acts to regulate genes known be important during the actual process of embryo attachment, endometrial remodeling, antioxidant defense mechanisms, and the modulation of the immune response around the implanting blastocyst.

Expression of LIF RNA and protein also appeared to be up-regulated by the action hCG. LIF has been shown to be essential for implantation in rodents (17). In human endometrium, LIF mRNA levels are low in the proliferative phase but rise dramatically in the mid to late luteal phase of the menstrual cycle (18, 19, 20). LIF immunoreactivity is detectable primarily in the glandular epithelium and the components of the LIF receptor complex (LIFR and gp130) are expressed throughout the menstrual cycle, mainly by luminal epithelium (19). Although it is not clear whether LIF plays an essential role in human implantation, there is now strong evidence that LIF also plays an important role in implantation in other primates. For example, in Rhesus monkeys, intrauterine injection of anti-LIF antibodies, on d 8 of pregnancy significantly reduces implantation rates (21). Our experiments have shown that the endometrial expression of LIF and its receptor gp130 are up-regulated in response to hCG treatment. LIF belongs to a group of related cytokines, which includes IL-6, IL-11, Cardiotrophin (CT-1), Oncostatin-M (OSM), Ciliary Neurotrophic Factor (CNTF), Cardiotrophin-like cytokine (CLC) and Neuropoietin (NP) (22). These molecules all share the gp130 protein as part of their signal transduction complex. Our microarray analysis failed to show up-regulation of IL-6 in baboon endometrium after hCG treatment. The hybridized signal intensity for IL-6 was above background in all arrays but due to large variance in the hCG-treated and control samples, IL-6 was not identified by our microarray analysis as up-regulated by hCG treatment. We examined IL-6 expression by RT-PCR and showed that as with LIF and gp130, IL-6 transcript expression is indeed up-regulated by hCG. Measurement of LIF and IL-6 protein in uterine flushings showed that this increase in transcript levels was mirrored by an increase in protein secretion into the uterine lumen. Interestingly, hCG has been shown to up-regulate LIF expression in human endometrium, both in experiments using an intrauterine microdialysis catheter (23) and after systemic treatment, using a luteotrophic regimen (24). It therefore appears that, in the baboon, CG from the preimplantation embryo is able to stimulate expression in the endometrium of molecules previously thought to be primarily regulated by steroids.

The up-regulation by hCG of the endometrial expression of C3 and C4A/B, suggests a role for hCG in the modulation of the periimplantation and decidual immune environment. C3 is integral to the activation of complement via the classical, alternative, and lectin activation pathways (25), with C4 being part of the classical activation pathway. The multiple actions of C3 allow it to promote phagocytosis, support local inflammatory responses to pathogens, and also to select appropriate antigens for the humoral response. Studies of endometrial Complement expression are limited, although in human endometrium, C3 has been localized to stromal and glandular compartments in the secretory phase of the menstrual cycle. The pattern of staining in the baboon endometrium in the d 10 PO controls was comparable with that reported in humans. However, in response to hCG, the stromal staining became predominant. The pattern of staining observed for C3 in the hCG-treated baboons also parallels the pattern of staining for the CG receptor in the baboon endometrium (9). In rodent uterus, however, C3 is up-regulated in epithelium by estrogen (26, 27, 28). Previous baboon studies have demonstrated increased glycodelin secretion, in response to intrauterine hCG infusion (29). The biological role of glycodelin, which is the most abundant secreted protein in early human pregnancy, is unclear, although in vitro immunosuppressive activity has been demonstrated (30). Taken together, the up-regulation of Complement C3, C4, and glycodelin expression suggests that hCG may orchestrate the endometrial immune adaptation to pregnancy.

A role for SOD, an enzyme that scavenges superoxide radicals, has been associated with endometrial function. Total SOD activity in human endometrium increases from the early proliferative phase and shows highest expression in the mid-secretory phase (31). Mice lacking SOD have reduced fertility (32), and SOD has been shown to play an important role in protecting the guinea pig uterus from oxidative damage during the window of receptivity (33). These studies suggest that uterine receptivity might be improved by enhancing antioxidant defenses. Also, in vitro decidualization of human endometrial stromal cells is associated with induction of the forkhead transcription factor FOXO1, which in turn can be shown to increase the expression of mitochondrial SOD2 (34). Interestingly, in contrast to undifferentiated stromal cells, decidualized HESC cells exposed to oxidative stress, show no increase in FOXO3, another member of the forkhead family and so appear to be inhibited from undergoing oxidative cell death. Taken together, these observations suggest that the differential regulation of FOXO proteins within decidua provides a mechanism for combating oxidative stress in pregnancy. Our finding that hCG significantly increases the expression of SOD suggests that, in preparation for implantation, the embryo also regulates an adaptive, endometrial response to oxidative stress.

Endometrial MMP expression has been previously shown to be steroidally regulated and is associated with tissue remodeling during menstruation (35, 36). MMP-23 is structurally different from other MMPs, and little is known of its biochemical function (37). Interestingly, MMP-23 is highly expressed in uterus and ovary, which given the cyclical, morphological changes that occur in these tissues, suggests a role in steroidally regulated matrix remodeling (37, 38). MMP-23 expression in immature rat uterus is confined to stroma, with expression by ovarian theca and granulosa cells (38). MMP-7 is a member of the stromelysin family of MMPs and degrades extracellular matrix proteins such as proteoglycans, fibronectin, elastin, and casein. MMP-7 is expressed in glandular epithelium (35, 39, 40), and in baboon and human endometrium the expression of MMP-7 is suppressed by progesterone (36, 41, 42). Up-regulation of MMP-7 expression in response to hCG has previously been demonstrated in periovulatory granulosa cells (43); however, our results show that hCG exerts a direct effect on peri-implantation endometrium. In the case of MMP-7, hCG is able to overcome the previously inhibitory effect of progesterone that is seen during the menstrual cycle.

SerpinA3 ({alpha}1 antichymotrypsin) is one of the most abundant plasma protease inhibitors and a member of the serine protease inhibitor class (44). The primary physiological function of SerpinA3 is to inhibit cathepsin G (45), a leukocyte-derived protease, which has been shown to cause epithelial detachment from basement membrane (46). Endometrial expression of SerpinA3 has been reported in stromal cells, with little epithelial expression (47). We speculate that hCG-induced expression of MMP-7, MMP-23, and SerpinA3 in stroma, contribute to cellular remodeling at the implantation site.

Secreted frizzled-related proteins are regulators of the Wnt signaling pathway and so modulate the functions of Wnt that include embryogenesis, cell proliferation, differentiation, and polarity, as well as cell adhesion and apoptosis and (48, 49). SFRP4 specifically inhibits Wnt-8 activity in Xenopus embryos (50) and SFRP4 expression has been shown to be down-regulated in human endometrium, during the secretory phase of the menstrual cycle (51). This down-regulation is accompanied by an increase in the expression of Dickkopf (Dkk-1), another inhibitor of Wnt signaling. The net biological impact of the changes in expression of SFRP4 and Dkk-1 in secretory phase endometrium may regulate the endometrial differentiation that is seen at the time of embryo attachment. We have shown the inhibition of SFRP4 expression by hCG, which may permit further stromal cell differentiation, to form the specialized decidua of pregnancy. Furthermore, it may also play a role in preventing apoptosis within the endometrium. Our recent studies have indicated that treatment with hCG or progesterone at the mid-luteal phase inhibits apoptosis in late secretory phase, implying a role for embryonic signals in the inhibition of apoptosis (52). SFRP4 expression is associated with apoptosis in the rodent corpus luteum, mammary gland, and ventral prostate (53, 54), although additional nonapoptotic functions for this molecule have also been reported (55). Thus, the decrease in SFRP4 in response to hCG in the endometrium may be a mechanism by which hCG prevents the normal course of endometrial apoptosis in the late secretory phase (56) in preparation for pregnancy. The expression of SFRP4 in pregnancy has not been studied in primates; however, in rodents, SFRP4 mRNA is detected in decidua only from d 9 of pregnancy and appears to be indirectly regulated by estrogen (57). The role of SFRP4 in late rodent pregnancy is unclear but may reflect a late phase of decidual cell proliferation.

Our experiments have shown that CG induces alterations in the expression of genes that regulate embryo attachment, endometrial remodeling, and the modulation of the immune response around the implanting blastocyst. These data support the concept that hCG acts not only to support the corpus luteum, but also plays and important role in endometrial adaptation to pregnancy. This indicates common mechanisms for early attachment events in both species. Our microarray analysis suggests that hCG may play several distinct roles in the endometrium, including remodeling, immunomodulation, and the novel finding that it up-regulates genes such as LIF that are known to play an important role in endometrial receptivity. It is known that the ability of blastocysts to produce hCG at specific stages of implantation is linked to pregnancy success (10), and therefore it is likely that hCG augmentation of endometrial expression of receptivity genes contributes to successful embryo apposition and attachment. In the past, it had been assumed that endometrial receptivity was under sole control of ovarian steroids. These finding suggest that in baboon at least, embryo-derived hCG may also be required for development of a fully receptive endometrium.


   Acknowledgments
 
We thank our colleagues in the microarray core facility, Department of Pathology, Cambridge University for microarrays and technical support.


   Footnotes
 
This study was supported by National Institutes of Health (NIH) Grants HD 36759 and HD 42280 (to A.T.F.). This facility is supported by the Biotechnology and Biological Sciences Research Council of the United Kingdom (BBSRC Grant No. 8/EGH16106). A.M.S. was supported by a Meres senior research fellowship from St. John’s College, Cambridge, and P.C. was supported by an Americas Fellowship supported by NIH National Institute of Child Health and Human Development D43TW00671.

Disclosure Statement: The authors have nothing to declare.

First Published Online November 16, 2006

Abbreviations: ads-cDNA, Amplified double-strand cDNA; C3, complement C3; CG, chorionic gonadotropin; hCG, human CG; IFN, interferon; IGFBP, IGF binding protein; LIF, leukemia inhibitory factor; PO, post ovulation; SDS, sodium dodecyl sulfate; SFRP4, Soluble Frizzled Related Protein-4; SOD, superoxide dismutase 2.

Received June 21, 2006.

Accepted for publication November 3, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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