Arch Gynecol Obstet DOI 10.1007/s00404-013-3080-9
GYNECOLOGIC ENDOCRINOLOGY AND REPRODUCTIVE MEDICINE
Modulation of tumor necrosis factor-stimulated gene-6 (TSG-6) expression in human endometrium Edison Capp • Caroline M. Milner • Joanna Williams • Lena Hauck • Julia Jauckus Thomas Strowitzki • Ariane Germeyer
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Received: 4 December 2012 / Accepted: 29 October 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Introduction The human endometrium undergoes repetitive, cyclical changes under the influence of endocrine signals (estrogen and progesterone). In particular, the extensive tissue remodeling, angiogenesis and leukocyte infiltration that occur during decidualization of the endometrium give rise to an environment that is permissive to blastocyst attachment. However, it is now well established that crosstalk (via paracrine signals) between the trophoblast and the endometrium is also a key for successful implantation. Microarray studies have identified TSG-6 as a gene with elevated expression in endometrial stromal cells following the exposure to trophoblast and immune cell products. TSG-6 is an inflammation-associated protein which acts as a potent inhibitor of neutrophil extravasation and also plays important roles in matrix remodeling, e.g., by promoting hyaluronan (HA) cross-linking and downregulating the protease network. Female TSG-6-/- mice are infertile and this has been attributed to their inability to E. Capp and C. M. Milner contributed equally. E. Capp L. Hauck J. Jauckus T. Strowitzki A. Germeyer (&) Department of Gynecological Endocrinology and Reproductive Medicine, University Hospital Heidelberg, INF440, 69120 Heidelberg, Germany e-mail: [email protected] E. Capp Department of Obstetrics and Gynecology, Hospital de Clinicas de Porto Alegre, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil C. M. Milner (&) J. Williams Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK e-mail: [email protected]
ovulate; however, the importance of TSG-6 in implantation has not been directly investigated. Material and methods Real-time PCR, as well as immunofluorescence staining was performed on endometrial biopsies of proliferative and secretory phase samples. In addition stromal cell cultures of human endometrium were used to assess the influence of different stimulating factors on the TSG-6 gene and protein expression via real-time PCR and ELISA. Results Herein demonstrate that TSG-6 mRNA is expressed by the endometrium during the proliferative and secretory phases of the menstrual cycle. We also show that conditioned media from placental tissues induce rapid upregulation of TSG-6 mRNA expression and sustained protein secretion, with evidence that TNF is an important factor in this effect. Furthermore, we demonstrate changes in protein expression in the mid-secretory phase in women affected by recurrent abortions. Conclusion These data suggest that TSG-6 expression might be essential in endometrial matrix organization and feto-maternal communication during the implantation process. Keywords Endometrium Placental tissue Recurrent abortion Paracrine regulation TSG-6
Introduction The human endometrium is a very plastic tissue undergoing repetitive regeneration and breakdown during the menstrual cycle, in response to ovarian hormones (reviewed in [1, 2]). High levels of estrogen in the pre-ovulatory (proliferative) phase promote cell proliferation and tissue re-growth. Postovulation, the secretory phase is characterized by elevated
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progesterone, which induces decidualization, such that the endometrium is receptive to blastocyst implantation. If embryo attachment and trophoblast invasion do not occur during the short ‘‘window of implantation’’, the superficial layer of the endometrium is shed. Decidualization is a complex process that involves differentiation of the endometrial stromal cells, influx of leukocytes [e.g., uterine natural killer (uNK) cells], extracellular matrix (ECM) remodeling and extensive angiogenesis. Studies of human gene expression and protein production (e.g., using microarrays) have provided important insights into the biological processes that underpin decidualization and subsequent implantation [3–6]. It has become clear that, in addition to the response of the endometrium to endocrine signals during the secretory phase, direct feto-maternal communication is essential for adequate implantation [7–9]. For example, in in vitro models, trophoblast-secreted products directly influence the gene expression profiles, and hence the activities, of endometrial stromal cells [10, 11]. TSG-6, the protein product of TNF-stimulated gene-6 (also known as TNFAIP6; TNF-induced protein-6) is up-regulated in endometrial stromal cells in response to trophoblastderived [10, 11] and immune cell factors [12]. TSG-6 is a secreted protein produced at sites of inflammation and tissue remodeling; its expression is highly upregulated by many cell types (e.g., smooth muscle cells, fibroblasts, epithelial cells and chondrocytes) in response to pro-inflammatory cytokines (e.g., TNF and IL-1) and, in some cases, by growth factors [13, 14]. TSG-6 has been detected in the context of inflammatory diseases, e.g., in the joints of arthritis patients [15, 16], and it protects against tissue damage in mouse models of arthritis [13, 17]. In this regard, TSG-6 is a potent inhibitor of neutrophil extravasation [18] and is regulating the protease network through its potentiation of the anti-plasmin activity of inter-ainhibitor (IaI) [19]. TSG-6 is also expressed during ovulation, by cumulus and granulosa cells within the ovarian follicle [20, 21], where it catalyzes the formation of covalent complexes between hyaluronan (HA) and the heavy chains (HC) of IaI [22, 23]. These HCHA complexes are essential for the correct organization of the HA-rich extracellular matrix (ECM) during expansion of the cumulus oocyte complex, such that female TSG-6-/- mice fail to ovulate fertilizable oocytes and are therefore infertile [22]. In recent microarray studies, TSG-6 gene expression was substantially up-regulated by endometrial cells both in response to trophoblast co-culture (i.e., a model of implantation) [11] and following incubation of decidualized endometrial stromal cells with trophoblast conditioned media [10]. In addition, a human microarray analysis of endometria of women with unexplained infertility compared to fertile controls at the time of implantation noted a
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diminished TSG-6 expression [7]. Therefore, TSG-6 might also contribute to uterine receptivity and blastocyst implantation, e.g., through the regulation of ECM remodeling, in addition to its role during ovulation [22]. The aim of this study was to assess the physiological changes in TSG-6 expression in human endometrial tissue during the late-proliferative and mid-secretory phases and its potential regulation in endometrial stromal cells by hormonal stimuli and/or placenta-derived factors. Furthermore, to determine its potential role during implantation, mid-secretory endometria of women with recurrent implantation failure were compared to endometria from healthy controls.
Materials and methods Patients Endometrial biopsies from the late-proliferative phase (day 8–12; n = 4) and LH-dated mid-secretory phase (day 20–24; n = 4) were collected for RNA analysis and six proliferative endometrial biopsies were collected for cell culture experiments. All biopsies were taken from healthy women with regular menstrual cycles, who were undergoing laparoscopic surgery or hysterectomy for benign reasons. Exclusion criteria were hormonal stimulation within the last 3 months, endocrinopathies, cancerous lesions and irregular menstrual bleeding. In addition, LH-dated midsecretory phase endometria were collected from women with recurrent spontaneous abortions (RSA; n = 4) and from healthy controls (n = 5) with no history of pregnancy complications. Recurrent spontaneous abortion was defined as more than two consecutive spontaneous abortions in the first trimester. None of these women gave birth. Histological examination of the endometrial biopsies was performed to confirm the appropriate phase, according to Noyes’ criteria [24]. Placental tissue (n = 12) was obtained following legal termination of uncomplicated pregnancies during the first trimester. All participants gave their informed consent for the use of samples under the approved Ethics protocol of the Ruprecht Karls University, Heidelberg. Cell isolation Stromal cells, epithelial cells and leukocytes were isolated by positive and negative selection with antibody-coated beads from late-proliferative (n = 4) and mid-secretory phase endometria (n = 3–4) essentially as described previously [25]. Briefly, endometrial tissue was digested at 37 °C for 1 h with 500 ll collagenase IV (5,000 U/ml, Sigma-Aldrich, Taufkirchen, Germany), 500 ll DNAse I (4 mg/ml, Roche Diagnostics GmbH, Mannheim, Germany), and 1 ml hyaluronidase IV (4,000 U/ml, Sigma-
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Aldrich) in 8 ml MCDB-105 (Sigma-Aldrich) and filtered through a 180-lm sieve. Further filtration, using a 40-lm sieve, resulted in entrapment of epithelial cells. Positive selection of leukocytes was carried out using DynabeadsÒ CD45 (Lot Nr. 11153D, DynalBiotech GmbH, Hamburg, Germany). Thereafter the remaining stromal cells were purified by negative selection using a bead mix of CELLectionTM Epithelial Enrich (Lot No. 16203), DynabeadsÒ CD31 Endothelial Cell (Lot Nr. 11155D), and DynabeadsÒ CD45 as described [25]. Finally stromal cells were cultured for 2 h to remove any remaining contaminating cells. Isolated cells were then snap frozen in TRIzol (Gibco, Karlsruhe, Germany) for subsequent mRNA isolation.
mincing into 5 mm3 pieces. After extensive washing in warm PBS to remove contaminating blood cells, the small pieces of tissue were incubated in DMEM enriched with estrogen (10 nM), progesterone (1 lM) and EGF (0.02 lg/ ml, Sigma-Aldrich). After 24 h (37 °C, 5 % CO2), this placenta-conditioned medium (PCM) was collected and cells were removed by centrifugation (10 min, 7009g). The PCM was then snap frozen in liquid nitrogen. For cell culture experiments four different pools of two to three PCM were used to increase volume and to reduce interassay variability. ELISA analysis (see below) of the PCM pools revealed that these contained little or no TSG-6 protein (data not shown).
Stromal cell culture
RNA isolation and real-time PCR
Stromal cells were isolated from proliferative phase endometrial tissues as described above and cultured as reported previously [26]. Briefly, cells were plated in DMEM (Gibco, Karlsruhe, Germany)/MCDB-105 media (3:1) with 10 % (v/v) fetal bovine serum (Perbio Science, Bonn, Germany), containing Penicillin–Streptomycin (CCpro GmbH, Neustadt, Germany), Gentamycin (Gibco) and Nystatin (Gibco) for 2 h (37 °C, 5 % CO2) after which non-adherent cells were removed. Cells were then cultured to confluency and passaged twice to ensure removal of contaminating cells. These non-decidualised stromal cells (n = 5), as well as cells decidualized in vitro (n =5) (see below), were cultured in the absence or presence of TNF (10 ng/mL, R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany), beta hCG (bhCG, 50 U/mL, SigmaAldrich), estrogen (10 nM, Sigma-Aldrich)/progesterone (1 lM, Sigma-Aldrich), or in PCM (see below) for 6, 24 h or 10 days. Then supernatants and cells were collected and snap frozen in liquid nitrogen for subsequent ELISA analysis and mRNA isolation, respectively.
RNA was isolated from cells that had been snap frozen in TRIzol, according to the manufacturer’s instructions. After determination of RNA concentration with the spectrophotometric system (Eppendorf AG, Hamburg, Germany) and verification of the RNA integrity via electrophoresis, 1 lg of total RNA per 20 ll reaction was used for reverse transcription (RT) via the 1st Strand cDNA Synthesis Kit for RT-PCR (Roche, Mannheim, Germany). Quantitative real-time PCR was performed using Taqman primers for TSG-6 (hs00200180_m1; Applied Biosystems, Darmstadt, Germany) and RPL0 (hs99999902_m1; Applied Biosystems) as a control housekeeping gene. Amplification was initiated with 10 min incubation at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C on a 7500 Fast Real-time PCR System (Applied Biosystems, Darmstadt, Germany) according to the manufacturer’s protocol. For statistical analyses, DCT values for TSG-6 expression were normalized relative to the CT values of the housekeeping gene RPL0. DDCT values were then calculated to determine the fold change in expression between treated and untreated samples.
Decidualization of endometrial stromal cells Confluent stromal cells (see above) were incubated in serum-free DMEM/MCDB-105 medium containing estrogen (10 nM), progesterone (1 lM), EGF (0.02 lg/ml), ascorbic acid (50 lg/ml, Sigma), apo-transferrin (10 lg/ ml, Sigma), as well as antibiotics for 12–14 days. Media was changed every 2–3 days. Decidualization was confirmed by measurement of prolactin in culture supernatants using a sandwich ELISA (Siemens Diagnostics, Eschborn, Germany), as described prior [27]. Preparation of placenta-conditioned medium (PCM) Placental tissue was dissected from legal first trimester abortions, as established [11], followed by mechanical
Enzyme-linked immunosorbent assay (ELISA) for TSG-6 Levels of TSG-6 protein in culture media were measured by ELISA, essentially as described in [28]. Briefly, 96-well Maxisorp plates were coated with a rat anti-human TSG-6 monoclonal antibody (A38.1) [29] and dilutions of recombinant human TSG-6 (for standard curves) or culture media, in 0.25 % (w/v) bovine serum albumin/PBS/0.05 % (v/v) Tween-20, were added (100 ll/well). Bound TSG-6 was detected using biotinylated goat anti-human TSG-6 antibody (R&D Systems), followed by Extravidin-Alkaline Phosphatase (Sigma) and Sigma Fast p-nitrophenyl phosphate; absorbance values at 405 nm were measured after *1 h. All conditions were set up in quadruplicate, samples
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from each donor were analyzed at least twice for each culture condition. Fluorescent staining Cryosections (8 lm) of mid-luteal phase biopsies were fixed with ice-cold acetone, blocked for 2 h with 5 % (v/v) goat serum, 5 % (w/v) BSA in PBS and then incubated with a TSG-6-specific anti-serum (RAH-1) [30] (or normal rabbit serum as control) followed by Alexa Fluor 555-conjugated anti-rabbit IgG (Invitrogen) and with biotinylated hyaluronan-binding (HA) protein (CosmoBio, CA, USA) followed by Alexa Fluor 488-conjugated streptavidin (Invitrogen); all reagents were diluted 1:1000 in PBS/5 % (w/v) BSA. Cell nuclei were stained with DAPI and slides were mounted with Vectashield. TSG-6-associated fluorescence was quantified using ImageJ software (http:// imagej.nih.gov/ij/), where three randomly selected regions (2,500 lm2) of both stroma and epithelium were analyzed to obtain mean fluorescence values for each cell type in each tissue section; values were normalized against controls where RAH-1 was substituted with normal rabbit serum. Statistical analysis Paired t tests were used for the statistical evaluation of changes in gene expression or protein secretion by treated cells compared to their respective controls, i.e., mRNA from cells cultured in DMEM for 6 h or media from cells cultured in DMEM for the equivalent time points (6, 24 h or 10 days). Unpaired t tests were used for analysis of fluorescent staining. P \ 0.05 was defined as statistically significant.
Results
Fig. 1 TSG-6 mRNA expression in human endometrium. TSG-6 mRNA expression was analyzed by real-time PCR in human endometrial biopsies of whole endometrium (n = 4 each) and isolated cell populations collected during the late-proliferative (black bars; n = 4) and mid-secretory (gray bars; n = 3–4) phases. TSG-6 expression is depicted as DCT value, normalized relative to RPL0 (mean ct = 17.16; SEM 0.43). Data are plotted as mean values ± SEM. Note that a lower DCT value reflects a higher gene expression level
exposure of either estrogen/progesterone (E2P4) or bhCG to physiological concentrations. In contrast, TSG-6 mRNA expression increased significantly after incubation with TNF for 6 h (8.5-fold), 24 h (8.3-fold) or 10 days (7.3fold) or with PCM for 6 h (22.5-fold) or 24 h (8.3-fold) (Fig. 2a). Non-decidualized endometrial stromal cells cultured in DMEM/MCDB-105 secreted very little TSG-6 protein (\0.5 ng/ml); this was unaffected by treatment with bhCG, and only mildly increased after 10 days estrogen/ progesterone exposure (*0.58 ng/ml vs. *0.24 ng/ml). In contrast, TSG-6 protein (n = 5) concentration in the media increased substantially over time in response to TNF (*1, *4.7 and *6.4 ng/ml after 6, 24 h and 10 days, respectively) and PCM (*4.8 and *7.9 ng/ml after 6 and 24 h, respectively) (Fig. 2b). Regulation of TSG-6 expression in decidualized endometrial stromal cells
TSG-6 mRNA expression in whole endometrium TSG-6 mRNA was detected in whole endometrium, with no significant difference between the levels of expression in the late-proliferative (n = 4) and mid-secretory (n = 4) phases (Fig. 1). Similar results were obtained for isolated cell populations, namely CD45? hematopoietic cells, epithelial cells and stromal cells (Fig. 1); this indicates that both non-decidualized and decidualized stromal cells express TSG-6 mRNA. Regulation of TSG-6 expression in non-decidualized endometrial stromal cells Cultured, non-decidualized stromal cells (n = 5) from proliferative phase endometrium expressed TSG-6 mRNA and the level of expression was essentially unaltered by
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Significant upregulation of TSG-6 mRNA expression (n = 5) was detected after incubation of decidualized endometrial stromal cells for 6 h with TNF (46.2-fold) or PCM (42.6-fold) (Fig. 2c). After 24 h incubation with TNF or PCM, TSG-6 mRNA levels were substantially lower than at the 6 h time points (9.7- and 7.4-fold upregulation compared to control, respectively). No effect was seen following exposure of these cells to bhCG. TSG-6 protein expression in decidualized cells (n = 4) significantly increased upon stimulation with TNF (*1.4 and *3.6 ng/ ml after 6 and 24 h, respectively) as well as PCM (*8.2 and *8.8 ng/ml after 6 and 24 h, respectively) (Fig. 2d). Thus, the observed upregulation of TSG-6 mRNA expression in endometrial stromal cells was translated into sustained protein production, followed by protein secretion into the media, as measured via ELISA.
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Fig. 2 TSG-6 mRNA (a, c) and protein (b, d) expression in nondecidualized (a, b) and decidualized (c, d) human endometrial stromal cells in vitro. Stromal cells were cultured in the absence or presence of estrogen (10 nM)/progesterone (1 nM) (E2/P4), bhCG (50 U/ml), TNF (10 ng/ml), or in PCM for the times indicated. TSG-6 mRNA expression was assessed by real-time PCR and the fold change
compared to control was calculated using the DDCT method. Concentrations of TSG-6 protein in the culture media were determined by ELISA and compared to controls at each of the times indicated. Data are plotted as mean values ± SEM. *p \ 0.05 and **p \ 0.01, as determined by paired t tests
Expression of TSG-6 protein in endometrial tissue
the late-proliferative and mid-secretory phases, suggesting that TSG-6 is not subject to endocrine regulation. This is consistent with the fact that TSG-6 has not been identified by microarray studies aimed at profiling changes in gene expression in the endometrium during the window of implantation [4, 31, 32]. However, reduced expression of TSG-6 (5-fold) has been reported in the endometria of women with unexplained infertility compared to fertile controls at the time of implantation (i.e., 7 days post-LH surge) [7], indicating that it might contribute to uterine receptivity. Our analysis of both non-decidualized and decidualized endometrial stromal cells in vitro revealed constitutive expression of TSG-6 mRNA and low levels of protein secretion (\0.5 ng/ml). The fact that TSG-6 expression was affected only minimally by prolonged exposure to estrogen/progesterone in vitro is in line with our observations for whole endometrium. However, elevated mRNA and protein levels in response to PCM indicate that TSG-6 expression by endometrial stromal cells is subject to paracrine regulation. Our findings confirm the previous microarray studies showing that TSG-6 gene expression is
Immunofluorescent staining of TSG-6 protein in endometrial biopsies taken at the time of implantation revealed significantly elevated levels of TSG-6 protein (p \ 0.01) associated with endometrial epithelial cells in women with RSA compared to controls. Stromal cells on the other hand showed only a marginal difference in TSG-6 expression between these two groups (Figs. 3, 4).
Discussion TSG-6 is known to be essential for female fertility in mice and this has been attributed to its role in cumulus matrix expansion during ovulation, where the nude oocytes of TSG-6-/- mice are resistant to fertilization in vivo [22]. However, the effect of TSG-6 gene deletion on blastocyst implantation has not been directly studied. We investigated TSG-6 expression in the human endometrium and showed that TSG-6 mRNA is present at similar levels in endometrial tissues (and isolated cell populations) collected during
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Fig. 3 Fluorescent staining of TSG-6 protein in endometrial biopsies. Cryosections (8 lm) of endometrial biopsies taken during the midsecretory phase from patients with RSA (a, b) and healthy controls with no history of complications during pregnancy (c, d) were stained for TSG-6 (using RAH-1 anti-serum) and HA (using HA-binding protein). Representative images are shown, where a and c illustrate grayscale data for TSG-6-associated fluorescence and b and d are the
corresponding merged color images showing the localizations of TSG-6 (red), HA (green) and cell nuclei (blue). No red fluorescence was detected in RSA or control tissues in experiments where RAH-1 was replaced by normal rabbit serum (not shown). Images were collected at 109 magnification and scale bars 100 lm. Examples of stroma and epithelium are indicated by asterisks and arrowheads, respectively
Fig. 4 Quantification of TSG-6 protein in endometrial tissue. TSG-6associated fluorescence (as illustrated in Fig. 3) was quantified using ImageJ in mid-secretory phase endometrial tissue from women with RSA (n = 4) and healthy controls with no history of complications during pregnancy (n = 5). Epithelial cells (a) and stromal cells (b) were evaluated separately for each patient and control; three independent measurements of fluorescence (within regions of
50 lm 9 50 lm) were recorded for each cell type and these were used to determine mean values. Data are presented as box and whisker plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes indicate the median values and the whiskers correspond to the 10th and 90th percentiles; outliers are shown as open circles. **p \ 0.01 and NS not significant, as determined by unpaired t tests
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up-regulated 4.4-fold in decidualized endometrial stromal cells following 24 h placental tissue co-culture [11] and 11- and 16-fold in decidualized cells incubated with PCM for 3 and 12 h, respectively [10]. In non-decidualized cells we observed a rapid upregulation in TSG-6 mRNA expression (28-fold) after just 6 h exposure to PCM that was substantially diminished after 24 h. In contrast, TNF induced a 10-fold increase in TSG-6 mRNA expression after 6 h that was sustained for up to 10 days. Both TNF and, to a greater extent, PCM promoted TSG-6 protein secretion that was detectable after 6 h and further elevated after 24 h (and 10 days in case of TNF). In decidualized cells, TNF and PCM had similar effects on TSG-6 mRNA production, where both induced rapid upregulation ([40fold) after 6 h that was much diminished after 24 h. The profiles of TSG-6 protein secretion by decidualized cells, in response to TNF or PCM, were very similar to those seen for non-decidualized cells both in terms of time dependency and protein levels. Together these results are consistent with the intimate feto-maternal communication that occurs at the site of implantation, where placental signals trigger a rapid and transient endometrial response. In this case, PCM promotes an increase in TSG-6 mRNA by decidualized endometrium, giving rise to sustained protein secretion, with accumulation of protein over at least a period of 6–24 h. Our data indicate that bhCG does not contribute to this PCM-mediated effect, whilst TNF might be an important contributor to the TSG-6 elevation noted. There is evidence for TNF expression by both trophoblast and decidualized endometrium during early pregnancy [33, 34] and this cytokine is known to induce the synthesis of TSG-6 mRNA and protein in many cell types, including fibroblasts [13]. The higher levels of TSG-6 protein produced in response to PCM compared to TNF indicate that other placenta-derived factors also play a role in up-regulating TSG-6 expression, while the identities of these remain to be established. TSG-6 mRNA was detected in whole endometrium and isolated stromal cells during both the proliferative and secretory phases and the culture media of both non-decidualized and decidualized stromal cells contained TSG-6 protein at \0.5 ng/ml. TSG-6 has been implicated as an endogenous regulator of inflammation; an effect that is likely mediated through multiple mechanisms, including down-regulation of protease activity and inhibition of neutrophil extravasation [16, 18, 19]. Immune and inflammatory processes are key components of decidualization, implantation and placentation and this is reflected by the gene expression profiles of endometrial cells [35– 37]. For example, the leukocyte population within the endometrium varies both in size and composition throughout the menstrual cycle and in early pregnancy [2, 12]. It is possible that elevated levels of TSG-6 during
implantation might contribute to the regulation of cell migration, for example, by modulating the expression or function of cytokines and chemokines in the endometrium and/or placental tissue. TSG-6 has also been implicated in ECM remodeling, both through direct cross-linking of HA [38] and via the catalysis of HCHA formation [23]. Deposition of HA in the human endometrium is elevated during the mid-proliferative and mid-secretory phases [39]; where the presence of TSG-6 could contribute to matrix reorganization in the context of proliferation, decidualization and implantation. This led us to examine TSG-6 expression in endometria from women with RSA in comparison to controls during the ‘‘window of implantation’’. We observed elevated TSG-6 protein expression in endometrial epithelial cells (i.e., the cells that make the first contact with the trophoblast), from women with RSA. This suggests that high levels of TSG-6 protein might contribute to an environment that is permissive to the implantation of otherwise incompetent trophoblast cells, i.e., as hypothesized by Salker et al. [40].
Conclusions TSG-6 mRNA was found to be expressed in the human endometrium in the late-proliferative and mid-secretory phases with only minimal expression change, indicating that TSG-6 is not subject to endocrine regulation, at least during these phases of the menstrual cycle. Nonetheless, the presence of TSG-6 in the endometrium suggests that it might be important in the maintenance of the ECM, e.g., via the formation of HCHA complexes. There was a rapid and transient upregulation in TSG-6 mRNA expression and sustained protein secretion by endometrial stromal cells in response to placenta-conditioned media, where this was mediated, at least in part, by TNF. This paracrine effect suggests a possible regulatory role for TSG-6 during blastocyst implantation, where tissue remodeling, leukocyte migration/activation and neo-vascularization need to be correctly controlled to ensure that the conceptus is securely attached to the wall of the uterus with an adequate blood supply. Last, but not least, the fact that women with RSA show elevated TSG-6 expression supports its importance in trophoblast implantation. Further studies to examine the role of TSG-6 in endometrium need to be performed. Acknowledgments The authors are grateful to Professor Tony Day and Dr. Roxana Popovici for helpful discussions and critical review of the manuscript. This work was supported by the Medical Research Council (Grant G0701180, CMM) and Arthritis Research UK (Grant 16539, CMM). Conflict of interest We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
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19. Mahoney DJ, Mulloy B, Forster MJ, Blundell CD, Fries E, Milner CM, Day AJ (2005) Characterization of the interaction between tumor necrosis factor-stimulated gene-6 and heparin: implications for the inhibition of plasmin in extracellular matrix microenvironments. J Biol Chem 280(29):27044–27055 20. Carrette O, Nemade RV, Day AJ, Brickner A, Larsen WJ (2001) TSG-6 is concentrated in the extracellular matrix of mouse cumulus oocyte complexes through hyaluronan and inter-alphainhibitor binding. Biol Reprod 65(1):301–308 21. Mukhopadhyay D, Hascall VC, Day AJ, Salustri A, Fulop C (2001) Two distinct populations of tumor necrosis factor-stimulated gene-6 protein in the extracellular matrix of expanded mouse cumulus cell-oocyte complexes. Arch Biochem Biophys 394(2):173–181 22. Fulop C, Szanto S, Mukhopadhyay D et al (2003) Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice. Development 130(10): 2253–2261 23. Rugg MS, Willis AC, Mukhopadhyay D, Hascall VC, Fries E, Fulop C, Milner CM, Day AJ (2005) Characterization of complexes formed between TSG-6 and inter-alpha-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan. J Biol Chem 280(27):25674–25686 24. Noyes RW, Hertig AT, Rock J (1950) Noyes RW, Hertig AT and Rock J. Dating the endometrial biopsy. Fertil Steril 1950; 1: 3-25. Fertil Steril 1:3–25 25. von Wolff M, Wang X, Gabius HJ, Strowitzki T (2005) Galectin fingerprinting in human endometrium and decidua during the menstrual cycle and in early gestation. Mol Hum Reprod 11(3):189–194 26. Germeyer A, Sharkey AM, Prasadajudio M et al (2009) Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts. Mol Hum Reprod 15(1):39–48 27. Germeyer A, Jauckus J, Zorn M, Toth B, Capp E, Strowitzki T (2011) Metformin modulates IL-8, IL-1beta, ICAM and IGFBP-1 expression in human endometrial stromal cells. Reprod Biomed Online 22(4):327–334 28. Maina V, Cotena A, Doni A, Nebuloni M, Pasqualini F, Milner CM, Day AJ, Mantovani A, Garlanda C (2009) Coregulation in human leukocytes of the long pentraxin PTX3 and TSG-6. J Leukoc Biol 86(1):123–132 29. Lesley J, English NM, Gal I, Mikecz K, Day AJ, Hyman R (2002) Hyaluronan binding properties of a CD44 chimera containing the link module of TSG-6. J Biol Chem 277(29):26600–26608 30. Fujimoto T, Savani RC, Watari M, Day AJ, Strauss JF 3rd (2002) Induction of the hyaluronic acid-binding protein, tumor necrosis factor-stimulated gene-6, in cervical smooth muscle cells by tumor necrosis factor-alpha and prostaglandin E(2). Am J Pathol 160(4):1495–1502 31. Carson DD, Lagow E, Thathiah A, Al-Shami R, Farach-Carson MC, Vernon M, Yuan L, Fritz MA, Lessey B (2002) Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Mol Hum Reprod 8(9):871–879 32. Riesewijk A, Martin J, van Os R, Horcajadas JA, Polman J, Pellicer A, Mosselman S, Simon C (2003) Gene expression profiling of human endometrial receptivity on days LH?2 versus LH?7 by microarray technology. Mol Hum Reprod 9(5):253–264 33. King A, Jokhi PP, Smith SK, Sharkey AM, Loke YW (1995) Screening for cytokine mRNA in human villous and extravillous trophoblasts using the reverse-transcriptase polymerase chain reaction (RT-PCR). Cytokine 7(4):364–371 34. Vince G, Shorter S, Starkey P, Humphreys J, Clover L, Wilkins T, Sargent I, Redman C (1992) Localization of tumour necrosis
Arch Gynecol Obstet factor production in cells at the materno/fetal interface in human pregnancy. Clin Exp Immunol 88(1):174–180 35. Dey SK, Lim H, Das SK, Reese J, Paria BC, Daikoku T, Wang H (2004) Molecular cues to implantation. Endocr Rev 25(3): 341–373 36. Dimitriadis E, White CA, Jones RL, Salamonsen LA (2005) Cytokines, chemokines and growth factors in endometrium related to implantation. Hum Reprod Update 11(6):613–630 37. Talbi S, Hamilton AE, Vo KC et al (2006) Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology 147(3):1097–1121
38. Baranova NS, Nileback E, Haller FM, Briggs DC, Svedhem S, Day AJ, Richter RP (2011) The inflammation-associated protein TSG-6 cross-links hyaluronan via hyaluronan-induced TSG-6 oligomers. J Biol Chem 286:25675–25686 39. Salamonsen LA, Shuster S, Stern R (2001) Distribution of hyaluronan in human endometrium across the menstrual cycle. Implications for implantation and menstruation. Cell Tissue Res 306(2):335–340 40. Salker M, Teklenburg G, Molokhia M et al (2010) Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss. PLoS ONE 5(4):e10287
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GYNECOLOGIC ENDOCRINOLOGY AND REPRODUCTIVE MEDICINE
Modulation of tumor necrosis factor-stimulated gene-6 (TSG-6) expression in human endometrium Edison Capp • Caroline M. Milner • Joanna Williams • Lena Hauck • Julia Jauckus Thomas Strowitzki • Ariane Germeyer
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Received: 4 December 2012 / Accepted: 29 October 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Introduction The human endometrium undergoes repetitive, cyclical changes under the influence of endocrine signals (estrogen and progesterone). In particular, the extensive tissue remodeling, angiogenesis and leukocyte infiltration that occur during decidualization of the endometrium give rise to an environment that is permissive to blastocyst attachment. However, it is now well established that crosstalk (via paracrine signals) between the trophoblast and the endometrium is also a key for successful implantation. Microarray studies have identified TSG-6 as a gene with elevated expression in endometrial stromal cells following the exposure to trophoblast and immune cell products. TSG-6 is an inflammation-associated protein which acts as a potent inhibitor of neutrophil extravasation and also plays important roles in matrix remodeling, e.g., by promoting hyaluronan (HA) cross-linking and downregulating the protease network. Female TSG-6-/- mice are infertile and this has been attributed to their inability to E. Capp and C. M. Milner contributed equally. E. Capp L. Hauck J. Jauckus T. Strowitzki A. Germeyer (&) Department of Gynecological Endocrinology and Reproductive Medicine, University Hospital Heidelberg, INF440, 69120 Heidelberg, Germany e-mail: [email protected] E. Capp Department of Obstetrics and Gynecology, Hospital de Clinicas de Porto Alegre, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil C. M. Milner (&) J. Williams Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK e-mail: [email protected]
ovulate; however, the importance of TSG-6 in implantation has not been directly investigated. Material and methods Real-time PCR, as well as immunofluorescence staining was performed on endometrial biopsies of proliferative and secretory phase samples. In addition stromal cell cultures of human endometrium were used to assess the influence of different stimulating factors on the TSG-6 gene and protein expression via real-time PCR and ELISA. Results Herein demonstrate that TSG-6 mRNA is expressed by the endometrium during the proliferative and secretory phases of the menstrual cycle. We also show that conditioned media from placental tissues induce rapid upregulation of TSG-6 mRNA expression and sustained protein secretion, with evidence that TNF is an important factor in this effect. Furthermore, we demonstrate changes in protein expression in the mid-secretory phase in women affected by recurrent abortions. Conclusion These data suggest that TSG-6 expression might be essential in endometrial matrix organization and feto-maternal communication during the implantation process. Keywords Endometrium Placental tissue Recurrent abortion Paracrine regulation TSG-6
Introduction The human endometrium is a very plastic tissue undergoing repetitive regeneration and breakdown during the menstrual cycle, in response to ovarian hormones (reviewed in [1, 2]). High levels of estrogen in the pre-ovulatory (proliferative) phase promote cell proliferation and tissue re-growth. Postovulation, the secretory phase is characterized by elevated
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progesterone, which induces decidualization, such that the endometrium is receptive to blastocyst implantation. If embryo attachment and trophoblast invasion do not occur during the short ‘‘window of implantation’’, the superficial layer of the endometrium is shed. Decidualization is a complex process that involves differentiation of the endometrial stromal cells, influx of leukocytes [e.g., uterine natural killer (uNK) cells], extracellular matrix (ECM) remodeling and extensive angiogenesis. Studies of human gene expression and protein production (e.g., using microarrays) have provided important insights into the biological processes that underpin decidualization and subsequent implantation [3–6]. It has become clear that, in addition to the response of the endometrium to endocrine signals during the secretory phase, direct feto-maternal communication is essential for adequate implantation [7–9]. For example, in in vitro models, trophoblast-secreted products directly influence the gene expression profiles, and hence the activities, of endometrial stromal cells [10, 11]. TSG-6, the protein product of TNF-stimulated gene-6 (also known as TNFAIP6; TNF-induced protein-6) is up-regulated in endometrial stromal cells in response to trophoblastderived [10, 11] and immune cell factors [12]. TSG-6 is a secreted protein produced at sites of inflammation and tissue remodeling; its expression is highly upregulated by many cell types (e.g., smooth muscle cells, fibroblasts, epithelial cells and chondrocytes) in response to pro-inflammatory cytokines (e.g., TNF and IL-1) and, in some cases, by growth factors [13, 14]. TSG-6 has been detected in the context of inflammatory diseases, e.g., in the joints of arthritis patients [15, 16], and it protects against tissue damage in mouse models of arthritis [13, 17]. In this regard, TSG-6 is a potent inhibitor of neutrophil extravasation [18] and is regulating the protease network through its potentiation of the anti-plasmin activity of inter-ainhibitor (IaI) [19]. TSG-6 is also expressed during ovulation, by cumulus and granulosa cells within the ovarian follicle [20, 21], where it catalyzes the formation of covalent complexes between hyaluronan (HA) and the heavy chains (HC) of IaI [22, 23]. These HCHA complexes are essential for the correct organization of the HA-rich extracellular matrix (ECM) during expansion of the cumulus oocyte complex, such that female TSG-6-/- mice fail to ovulate fertilizable oocytes and are therefore infertile [22]. In recent microarray studies, TSG-6 gene expression was substantially up-regulated by endometrial cells both in response to trophoblast co-culture (i.e., a model of implantation) [11] and following incubation of decidualized endometrial stromal cells with trophoblast conditioned media [10]. In addition, a human microarray analysis of endometria of women with unexplained infertility compared to fertile controls at the time of implantation noted a
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diminished TSG-6 expression [7]. Therefore, TSG-6 might also contribute to uterine receptivity and blastocyst implantation, e.g., through the regulation of ECM remodeling, in addition to its role during ovulation [22]. The aim of this study was to assess the physiological changes in TSG-6 expression in human endometrial tissue during the late-proliferative and mid-secretory phases and its potential regulation in endometrial stromal cells by hormonal stimuli and/or placenta-derived factors. Furthermore, to determine its potential role during implantation, mid-secretory endometria of women with recurrent implantation failure were compared to endometria from healthy controls.
Materials and methods Patients Endometrial biopsies from the late-proliferative phase (day 8–12; n = 4) and LH-dated mid-secretory phase (day 20–24; n = 4) were collected for RNA analysis and six proliferative endometrial biopsies were collected for cell culture experiments. All biopsies were taken from healthy women with regular menstrual cycles, who were undergoing laparoscopic surgery or hysterectomy for benign reasons. Exclusion criteria were hormonal stimulation within the last 3 months, endocrinopathies, cancerous lesions and irregular menstrual bleeding. In addition, LH-dated midsecretory phase endometria were collected from women with recurrent spontaneous abortions (RSA; n = 4) and from healthy controls (n = 5) with no history of pregnancy complications. Recurrent spontaneous abortion was defined as more than two consecutive spontaneous abortions in the first trimester. None of these women gave birth. Histological examination of the endometrial biopsies was performed to confirm the appropriate phase, according to Noyes’ criteria [24]. Placental tissue (n = 12) was obtained following legal termination of uncomplicated pregnancies during the first trimester. All participants gave their informed consent for the use of samples under the approved Ethics protocol of the Ruprecht Karls University, Heidelberg. Cell isolation Stromal cells, epithelial cells and leukocytes were isolated by positive and negative selection with antibody-coated beads from late-proliferative (n = 4) and mid-secretory phase endometria (n = 3–4) essentially as described previously [25]. Briefly, endometrial tissue was digested at 37 °C for 1 h with 500 ll collagenase IV (5,000 U/ml, Sigma-Aldrich, Taufkirchen, Germany), 500 ll DNAse I (4 mg/ml, Roche Diagnostics GmbH, Mannheim, Germany), and 1 ml hyaluronidase IV (4,000 U/ml, Sigma-
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Aldrich) in 8 ml MCDB-105 (Sigma-Aldrich) and filtered through a 180-lm sieve. Further filtration, using a 40-lm sieve, resulted in entrapment of epithelial cells. Positive selection of leukocytes was carried out using DynabeadsÒ CD45 (Lot Nr. 11153D, DynalBiotech GmbH, Hamburg, Germany). Thereafter the remaining stromal cells were purified by negative selection using a bead mix of CELLectionTM Epithelial Enrich (Lot No. 16203), DynabeadsÒ CD31 Endothelial Cell (Lot Nr. 11155D), and DynabeadsÒ CD45 as described [25]. Finally stromal cells were cultured for 2 h to remove any remaining contaminating cells. Isolated cells were then snap frozen in TRIzol (Gibco, Karlsruhe, Germany) for subsequent mRNA isolation.
mincing into 5 mm3 pieces. After extensive washing in warm PBS to remove contaminating blood cells, the small pieces of tissue were incubated in DMEM enriched with estrogen (10 nM), progesterone (1 lM) and EGF (0.02 lg/ ml, Sigma-Aldrich). After 24 h (37 °C, 5 % CO2), this placenta-conditioned medium (PCM) was collected and cells were removed by centrifugation (10 min, 7009g). The PCM was then snap frozen in liquid nitrogen. For cell culture experiments four different pools of two to three PCM were used to increase volume and to reduce interassay variability. ELISA analysis (see below) of the PCM pools revealed that these contained little or no TSG-6 protein (data not shown).
Stromal cell culture
RNA isolation and real-time PCR
Stromal cells were isolated from proliferative phase endometrial tissues as described above and cultured as reported previously [26]. Briefly, cells were plated in DMEM (Gibco, Karlsruhe, Germany)/MCDB-105 media (3:1) with 10 % (v/v) fetal bovine serum (Perbio Science, Bonn, Germany), containing Penicillin–Streptomycin (CCpro GmbH, Neustadt, Germany), Gentamycin (Gibco) and Nystatin (Gibco) for 2 h (37 °C, 5 % CO2) after which non-adherent cells were removed. Cells were then cultured to confluency and passaged twice to ensure removal of contaminating cells. These non-decidualised stromal cells (n = 5), as well as cells decidualized in vitro (n =5) (see below), were cultured in the absence or presence of TNF (10 ng/mL, R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany), beta hCG (bhCG, 50 U/mL, SigmaAldrich), estrogen (10 nM, Sigma-Aldrich)/progesterone (1 lM, Sigma-Aldrich), or in PCM (see below) for 6, 24 h or 10 days. Then supernatants and cells were collected and snap frozen in liquid nitrogen for subsequent ELISA analysis and mRNA isolation, respectively.
RNA was isolated from cells that had been snap frozen in TRIzol, according to the manufacturer’s instructions. After determination of RNA concentration with the spectrophotometric system (Eppendorf AG, Hamburg, Germany) and verification of the RNA integrity via electrophoresis, 1 lg of total RNA per 20 ll reaction was used for reverse transcription (RT) via the 1st Strand cDNA Synthesis Kit for RT-PCR (Roche, Mannheim, Germany). Quantitative real-time PCR was performed using Taqman primers for TSG-6 (hs00200180_m1; Applied Biosystems, Darmstadt, Germany) and RPL0 (hs99999902_m1; Applied Biosystems) as a control housekeeping gene. Amplification was initiated with 10 min incubation at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C on a 7500 Fast Real-time PCR System (Applied Biosystems, Darmstadt, Germany) according to the manufacturer’s protocol. For statistical analyses, DCT values for TSG-6 expression were normalized relative to the CT values of the housekeeping gene RPL0. DDCT values were then calculated to determine the fold change in expression between treated and untreated samples.
Decidualization of endometrial stromal cells Confluent stromal cells (see above) were incubated in serum-free DMEM/MCDB-105 medium containing estrogen (10 nM), progesterone (1 lM), EGF (0.02 lg/ml), ascorbic acid (50 lg/ml, Sigma), apo-transferrin (10 lg/ ml, Sigma), as well as antibiotics for 12–14 days. Media was changed every 2–3 days. Decidualization was confirmed by measurement of prolactin in culture supernatants using a sandwich ELISA (Siemens Diagnostics, Eschborn, Germany), as described prior [27]. Preparation of placenta-conditioned medium (PCM) Placental tissue was dissected from legal first trimester abortions, as established [11], followed by mechanical
Enzyme-linked immunosorbent assay (ELISA) for TSG-6 Levels of TSG-6 protein in culture media were measured by ELISA, essentially as described in [28]. Briefly, 96-well Maxisorp plates were coated with a rat anti-human TSG-6 monoclonal antibody (A38.1) [29] and dilutions of recombinant human TSG-6 (for standard curves) or culture media, in 0.25 % (w/v) bovine serum albumin/PBS/0.05 % (v/v) Tween-20, were added (100 ll/well). Bound TSG-6 was detected using biotinylated goat anti-human TSG-6 antibody (R&D Systems), followed by Extravidin-Alkaline Phosphatase (Sigma) and Sigma Fast p-nitrophenyl phosphate; absorbance values at 405 nm were measured after *1 h. All conditions were set up in quadruplicate, samples
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from each donor were analyzed at least twice for each culture condition. Fluorescent staining Cryosections (8 lm) of mid-luteal phase biopsies were fixed with ice-cold acetone, blocked for 2 h with 5 % (v/v) goat serum, 5 % (w/v) BSA in PBS and then incubated with a TSG-6-specific anti-serum (RAH-1) [30] (or normal rabbit serum as control) followed by Alexa Fluor 555-conjugated anti-rabbit IgG (Invitrogen) and with biotinylated hyaluronan-binding (HA) protein (CosmoBio, CA, USA) followed by Alexa Fluor 488-conjugated streptavidin (Invitrogen); all reagents were diluted 1:1000 in PBS/5 % (w/v) BSA. Cell nuclei were stained with DAPI and slides were mounted with Vectashield. TSG-6-associated fluorescence was quantified using ImageJ software (http:// imagej.nih.gov/ij/), where three randomly selected regions (2,500 lm2) of both stroma and epithelium were analyzed to obtain mean fluorescence values for each cell type in each tissue section; values were normalized against controls where RAH-1 was substituted with normal rabbit serum. Statistical analysis Paired t tests were used for the statistical evaluation of changes in gene expression or protein secretion by treated cells compared to their respective controls, i.e., mRNA from cells cultured in DMEM for 6 h or media from cells cultured in DMEM for the equivalent time points (6, 24 h or 10 days). Unpaired t tests were used for analysis of fluorescent staining. P \ 0.05 was defined as statistically significant.
Results
Fig. 1 TSG-6 mRNA expression in human endometrium. TSG-6 mRNA expression was analyzed by real-time PCR in human endometrial biopsies of whole endometrium (n = 4 each) and isolated cell populations collected during the late-proliferative (black bars; n = 4) and mid-secretory (gray bars; n = 3–4) phases. TSG-6 expression is depicted as DCT value, normalized relative to RPL0 (mean ct = 17.16; SEM 0.43). Data are plotted as mean values ± SEM. Note that a lower DCT value reflects a higher gene expression level
exposure of either estrogen/progesterone (E2P4) or bhCG to physiological concentrations. In contrast, TSG-6 mRNA expression increased significantly after incubation with TNF for 6 h (8.5-fold), 24 h (8.3-fold) or 10 days (7.3fold) or with PCM for 6 h (22.5-fold) or 24 h (8.3-fold) (Fig. 2a). Non-decidualized endometrial stromal cells cultured in DMEM/MCDB-105 secreted very little TSG-6 protein (\0.5 ng/ml); this was unaffected by treatment with bhCG, and only mildly increased after 10 days estrogen/ progesterone exposure (*0.58 ng/ml vs. *0.24 ng/ml). In contrast, TSG-6 protein (n = 5) concentration in the media increased substantially over time in response to TNF (*1, *4.7 and *6.4 ng/ml after 6, 24 h and 10 days, respectively) and PCM (*4.8 and *7.9 ng/ml after 6 and 24 h, respectively) (Fig. 2b). Regulation of TSG-6 expression in decidualized endometrial stromal cells
TSG-6 mRNA expression in whole endometrium TSG-6 mRNA was detected in whole endometrium, with no significant difference between the levels of expression in the late-proliferative (n = 4) and mid-secretory (n = 4) phases (Fig. 1). Similar results were obtained for isolated cell populations, namely CD45? hematopoietic cells, epithelial cells and stromal cells (Fig. 1); this indicates that both non-decidualized and decidualized stromal cells express TSG-6 mRNA. Regulation of TSG-6 expression in non-decidualized endometrial stromal cells Cultured, non-decidualized stromal cells (n = 5) from proliferative phase endometrium expressed TSG-6 mRNA and the level of expression was essentially unaltered by
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Significant upregulation of TSG-6 mRNA expression (n = 5) was detected after incubation of decidualized endometrial stromal cells for 6 h with TNF (46.2-fold) or PCM (42.6-fold) (Fig. 2c). After 24 h incubation with TNF or PCM, TSG-6 mRNA levels were substantially lower than at the 6 h time points (9.7- and 7.4-fold upregulation compared to control, respectively). No effect was seen following exposure of these cells to bhCG. TSG-6 protein expression in decidualized cells (n = 4) significantly increased upon stimulation with TNF (*1.4 and *3.6 ng/ ml after 6 and 24 h, respectively) as well as PCM (*8.2 and *8.8 ng/ml after 6 and 24 h, respectively) (Fig. 2d). Thus, the observed upregulation of TSG-6 mRNA expression in endometrial stromal cells was translated into sustained protein production, followed by protein secretion into the media, as measured via ELISA.
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Fig. 2 TSG-6 mRNA (a, c) and protein (b, d) expression in nondecidualized (a, b) and decidualized (c, d) human endometrial stromal cells in vitro. Stromal cells were cultured in the absence or presence of estrogen (10 nM)/progesterone (1 nM) (E2/P4), bhCG (50 U/ml), TNF (10 ng/ml), or in PCM for the times indicated. TSG-6 mRNA expression was assessed by real-time PCR and the fold change
compared to control was calculated using the DDCT method. Concentrations of TSG-6 protein in the culture media were determined by ELISA and compared to controls at each of the times indicated. Data are plotted as mean values ± SEM. *p \ 0.05 and **p \ 0.01, as determined by paired t tests
Expression of TSG-6 protein in endometrial tissue
the late-proliferative and mid-secretory phases, suggesting that TSG-6 is not subject to endocrine regulation. This is consistent with the fact that TSG-6 has not been identified by microarray studies aimed at profiling changes in gene expression in the endometrium during the window of implantation [4, 31, 32]. However, reduced expression of TSG-6 (5-fold) has been reported in the endometria of women with unexplained infertility compared to fertile controls at the time of implantation (i.e., 7 days post-LH surge) [7], indicating that it might contribute to uterine receptivity. Our analysis of both non-decidualized and decidualized endometrial stromal cells in vitro revealed constitutive expression of TSG-6 mRNA and low levels of protein secretion (\0.5 ng/ml). The fact that TSG-6 expression was affected only minimally by prolonged exposure to estrogen/progesterone in vitro is in line with our observations for whole endometrium. However, elevated mRNA and protein levels in response to PCM indicate that TSG-6 expression by endometrial stromal cells is subject to paracrine regulation. Our findings confirm the previous microarray studies showing that TSG-6 gene expression is
Immunofluorescent staining of TSG-6 protein in endometrial biopsies taken at the time of implantation revealed significantly elevated levels of TSG-6 protein (p \ 0.01) associated with endometrial epithelial cells in women with RSA compared to controls. Stromal cells on the other hand showed only a marginal difference in TSG-6 expression between these two groups (Figs. 3, 4).
Discussion TSG-6 is known to be essential for female fertility in mice and this has been attributed to its role in cumulus matrix expansion during ovulation, where the nude oocytes of TSG-6-/- mice are resistant to fertilization in vivo [22]. However, the effect of TSG-6 gene deletion on blastocyst implantation has not been directly studied. We investigated TSG-6 expression in the human endometrium and showed that TSG-6 mRNA is present at similar levels in endometrial tissues (and isolated cell populations) collected during
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Fig. 3 Fluorescent staining of TSG-6 protein in endometrial biopsies. Cryosections (8 lm) of endometrial biopsies taken during the midsecretory phase from patients with RSA (a, b) and healthy controls with no history of complications during pregnancy (c, d) were stained for TSG-6 (using RAH-1 anti-serum) and HA (using HA-binding protein). Representative images are shown, where a and c illustrate grayscale data for TSG-6-associated fluorescence and b and d are the
corresponding merged color images showing the localizations of TSG-6 (red), HA (green) and cell nuclei (blue). No red fluorescence was detected in RSA or control tissues in experiments where RAH-1 was replaced by normal rabbit serum (not shown). Images were collected at 109 magnification and scale bars 100 lm. Examples of stroma and epithelium are indicated by asterisks and arrowheads, respectively
Fig. 4 Quantification of TSG-6 protein in endometrial tissue. TSG-6associated fluorescence (as illustrated in Fig. 3) was quantified using ImageJ in mid-secretory phase endometrial tissue from women with RSA (n = 4) and healthy controls with no history of complications during pregnancy (n = 5). Epithelial cells (a) and stromal cells (b) were evaluated separately for each patient and control; three independent measurements of fluorescence (within regions of
50 lm 9 50 lm) were recorded for each cell type and these were used to determine mean values. Data are presented as box and whisker plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes indicate the median values and the whiskers correspond to the 10th and 90th percentiles; outliers are shown as open circles. **p \ 0.01 and NS not significant, as determined by unpaired t tests
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up-regulated 4.4-fold in decidualized endometrial stromal cells following 24 h placental tissue co-culture [11] and 11- and 16-fold in decidualized cells incubated with PCM for 3 and 12 h, respectively [10]. In non-decidualized cells we observed a rapid upregulation in TSG-6 mRNA expression (28-fold) after just 6 h exposure to PCM that was substantially diminished after 24 h. In contrast, TNF induced a 10-fold increase in TSG-6 mRNA expression after 6 h that was sustained for up to 10 days. Both TNF and, to a greater extent, PCM promoted TSG-6 protein secretion that was detectable after 6 h and further elevated after 24 h (and 10 days in case of TNF). In decidualized cells, TNF and PCM had similar effects on TSG-6 mRNA production, where both induced rapid upregulation ([40fold) after 6 h that was much diminished after 24 h. The profiles of TSG-6 protein secretion by decidualized cells, in response to TNF or PCM, were very similar to those seen for non-decidualized cells both in terms of time dependency and protein levels. Together these results are consistent with the intimate feto-maternal communication that occurs at the site of implantation, where placental signals trigger a rapid and transient endometrial response. In this case, PCM promotes an increase in TSG-6 mRNA by decidualized endometrium, giving rise to sustained protein secretion, with accumulation of protein over at least a period of 6–24 h. Our data indicate that bhCG does not contribute to this PCM-mediated effect, whilst TNF might be an important contributor to the TSG-6 elevation noted. There is evidence for TNF expression by both trophoblast and decidualized endometrium during early pregnancy [33, 34] and this cytokine is known to induce the synthesis of TSG-6 mRNA and protein in many cell types, including fibroblasts [13]. The higher levels of TSG-6 protein produced in response to PCM compared to TNF indicate that other placenta-derived factors also play a role in up-regulating TSG-6 expression, while the identities of these remain to be established. TSG-6 mRNA was detected in whole endometrium and isolated stromal cells during both the proliferative and secretory phases and the culture media of both non-decidualized and decidualized stromal cells contained TSG-6 protein at \0.5 ng/ml. TSG-6 has been implicated as an endogenous regulator of inflammation; an effect that is likely mediated through multiple mechanisms, including down-regulation of protease activity and inhibition of neutrophil extravasation [16, 18, 19]. Immune and inflammatory processes are key components of decidualization, implantation and placentation and this is reflected by the gene expression profiles of endometrial cells [35– 37]. For example, the leukocyte population within the endometrium varies both in size and composition throughout the menstrual cycle and in early pregnancy [2, 12]. It is possible that elevated levels of TSG-6 during
implantation might contribute to the regulation of cell migration, for example, by modulating the expression or function of cytokines and chemokines in the endometrium and/or placental tissue. TSG-6 has also been implicated in ECM remodeling, both through direct cross-linking of HA [38] and via the catalysis of HCHA formation [23]. Deposition of HA in the human endometrium is elevated during the mid-proliferative and mid-secretory phases [39]; where the presence of TSG-6 could contribute to matrix reorganization in the context of proliferation, decidualization and implantation. This led us to examine TSG-6 expression in endometria from women with RSA in comparison to controls during the ‘‘window of implantation’’. We observed elevated TSG-6 protein expression in endometrial epithelial cells (i.e., the cells that make the first contact with the trophoblast), from women with RSA. This suggests that high levels of TSG-6 protein might contribute to an environment that is permissive to the implantation of otherwise incompetent trophoblast cells, i.e., as hypothesized by Salker et al. [40].
Conclusions TSG-6 mRNA was found to be expressed in the human endometrium in the late-proliferative and mid-secretory phases with only minimal expression change, indicating that TSG-6 is not subject to endocrine regulation, at least during these phases of the menstrual cycle. Nonetheless, the presence of TSG-6 in the endometrium suggests that it might be important in the maintenance of the ECM, e.g., via the formation of HCHA complexes. There was a rapid and transient upregulation in TSG-6 mRNA expression and sustained protein secretion by endometrial stromal cells in response to placenta-conditioned media, where this was mediated, at least in part, by TNF. This paracrine effect suggests a possible regulatory role for TSG-6 during blastocyst implantation, where tissue remodeling, leukocyte migration/activation and neo-vascularization need to be correctly controlled to ensure that the conceptus is securely attached to the wall of the uterus with an adequate blood supply. Last, but not least, the fact that women with RSA show elevated TSG-6 expression supports its importance in trophoblast implantation. Further studies to examine the role of TSG-6 in endometrium need to be performed. Acknowledgments The authors are grateful to Professor Tony Day and Dr. Roxana Popovici for helpful discussions and critical review of the manuscript. This work was supported by the Medical Research Council (Grant G0701180, CMM) and Arthritis Research UK (Grant 16539, CMM). Conflict of interest We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
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