Differential regulation of osteopontin and CD44 correlates with infertility status in PCOS patients.

Endometrial receptivity is mediated by adhesion molecules at the endometrium-trophoblast interface where osteopontin (OPN) and CD44 form a protein complex that plays an important role in embryo recognition. Here, we undertook a prospective study investigating the expression and regulation of OPN and CD44 in 50 fertile and 31 infertile ovulatory polycystic ovarian syndrome (PCOS) patients in the proliferative and secretory phases of the natural menstrual cycle and in 12 infertile anovulatory PCOS patients. Endometrial biopsies and blood samples were evaluated for expression of OPN and CD44 using RT-PCR, immunohistochemistry and ELISA analysis to determine circulating levels of OPN, CD44, TNF-α, IFN-γ and OPN and CD44 levels in biopsy media. Our findings highlighted an increased level of circulating OPN and CD44 in serum from infertile patients that inversely correlated with expression levels in endometrial tissue and positively correlated with levels secreted into biopsy media. OPN and CD44 levels positively correlated to each other in serum and media from fertile and PCOS patients, as well as to circulating TNF-α and IFN-γ. In vitro analysis revealed that hormone treatment induced recruitment of ERα to the OPN and CD44 promoters with a concomitant increase in the expression of these genes. In infertile patients, inflammatory cytokines led to recruitment of NF-κB and STAT1 proteins to the OPN and CD44 promoters, resulting in their overexpression. These observations suggest that the endometrial epithelial OPN-CD44 adhesion complex is deficient in ovulatory PCOS patients and displays an altered stoichiometry in anovulatory patients, which in both cases may perturb apposition. This, together with elevated circulating and local secreted levels of these proteins, may hinder endometrium-trophoblast interactions by saturating OPN and CD44 receptors on the surface of the blastocyst, thereby contributing to the infertility associated with ovulating PCOS patients. KEY MESSAGES: • Endometrial epithelial OPN-CD44 adhesion complex levels are deficient in ovulatory PCOS patients contributing to the endometrial infertility associated with ovulating PCOS patients. • Circulating levels of OPN, CD44 and inflammatory cytokines TNF-α and IFN-γ are altered in infertile PCOS patients. • Increased levels of both OPN and CD44 in biopsy media and serum inversely correlate with endometrial expression of these markers in endometrial tissue. • In infertile PCOS patients, high levels of oestrogens and inflammatory cytokines stimulate the recruitment of transcription factors to the OPN and CD44 promoters to enhance gene transcription. • Our study identifies a novel crosstalk between the CD44-OPN adhesion complex, ERα, STAT1 and NF-κB pathways modulating endometrial receptivity.

Cu isotope ratios are meaningful in ovarian cancer diagnosis.

BACKGROUND: Ovarian cancer diagnosis is currently based on imaging and circulating CA-125 concentrations with well-known limits to sensitivity and specificity. New biomarkers are required to complement CA-125 testing to increase effectiveness. Increases in sensitivity of isotopic separation via multi collector inductively coupled plasma-mass spectrometry have recently allowed highly accurate measurement of copper (Cu) isotopic variations. Studies in breast cancer patients have revealed changes of serum copper isotopic composition demonstrating the potential for development as a cancer biomarker. Evaluating 65Cu/63Cu ratios (δ65Cu) in serum samples from cancer patients has revealed a strong correlation with cancer development. In this study blood samples from forty-four ovarian cancer patients, and 13 ovarian biopsies were investigated. RESULTS: Here we demonstrate that changes in Cu isotopes also occurs in ovarian cancer patients. Copper composition determined by multiple collector inductively coupled plasma mass spectrometry revealed that the copper isotopic ratio δ65Cu in the plasma of 44 ovarian cancer patient cohort was significantly lower than in a group of 48 healthy donors, and indicated that serum was enriched for 63Cu. Further analysis revealed that the isotopic composition of tumour biopsies was enriched for 65Cu compared with adjacent healthy ovarian tissues. CONCLUSIONS: We propose that these changes are due to increase lactate and Cu transporter activities in the tumour. These observations demonstrate that, combined with existing strategies, δ65Cu could be developed for use in ovarian cancer early detection.

POISED-5, a portable on-board electrochemical impedance spectroscopy biomarker analysis device.

Point-of-care medical devices offer the potential for rapid biomarker detection and reporting of medical conditions, thereby bypassing the requirements for offline clinical laboratory facilities in many cases. Label-free electrochemical techniques are suitable for use in handheld diagnostic devices due the inherent electronic detection modality and low requirement for processing reagents. While electrochemical impedance sensing is widely used in tissue analysis such as body composition measurement, its use in point-of-care patient testing is yet to be widely adopted. Here we have considered a number of issues currently limiting the translation of electrochemical impedance sensing into clinical biosensor devices. Specifically, we have addressed the current requirement for these sensors to be connected to an external processor by applying a minimum number of frequencies required for optimized biomarker detection, and subsequently delivering analytics within the measurement device. The POISED-5 device was evaluated using a sensor for the ovarian cancer biomarker cancer antigen 125 (CA125), demonstrating performance comparable to standard laboratory equipment, with direct interpretation of response signal amplitude substituting traditional impedance component calculation and model fitting.

Direct monitoring of breast and endometrial cancer cell epigenetic response to DNA methyltransferase and histone deacetylase inhibitors.

DNA methylation and histone deacetylation are key epigenetic processes involved in normal cellular function and tumorigenesis. Therapeutic strategies based on DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors are currently in use and under development for the treatment of cancers. Genome-wide DNA methylation profiling has been proposed for use in disease diagnosis, and histone modification profiling for disease stratification will follow suit. However, whether epigenome sequencing technologies will be feasible for rapid clinic diagnosis and patient treatment monitoring remains to be seen, and alternative detection technologies will almost certainly be needed. Here we used electrochemical impedance spectroscopy (EIS) employing a graphene-based screen-printed electrode system to directly measure global DNA methylation and histone H3 acetylation to compare non-cancer and breast cancer cell lines. We demonstrated that whilst global methylation was not useful as a differential marker in the cellular systems tested, histone H3 acetylation was effective at higher chromatin levels. Using breast and endometrial cancer cell models, EIS was then used to monitor cellular responses to the DNMT and HDAC inhibitors 5-Aza-2'-deoxycytidine and suberoylanilide hydroxamic acid in vitro, and proved very effective at detecting global cellular responses to either treatment, indicating that this approach could be useful in following treatment response to epigenetic drugs. Moreover, this work reports the first combined analysis of two epigenetic markers using a unified graphene-based biosensor platform, demonstrating the potential for multiplex analysis of both methylation and acetylation on the same sample.

Sub-ppm level high energy resolution fluorescence detected X-ray absorption spectroscopy of selenium in articular cartilage.

The speciation of highly-diluted elements by X-ray absorption spectroscopy in a diverse range of materials is extremely challenging, especially in biological matrices such as articular cartilage. Here we show that using a high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) technique coupled to an array of crystal analyzers, selenium speciation down to 400 ppb (µg kg-1) within articular cartilage can be demonstrated. This is a major advance in the speciation of highly-diluted elements through X-ray absorption spectroscopy and opens new possibilities to study the metabolic role of selenium and other elements in biological samples.

A three-dimensional model of primary bovine endometrium using an electrospun scaffold.

Endometrial stromal and epithelial cell function is typically studied in vitro using standard two-dimensional monocultures, but these cultures fail to reflect the complex three-dimensional (3D) architecture of tissue. A 3D model of bovine endometrium that reflects the architectural arrangement of in vivo tissue would beneficially assist the study of tissue function. An electrospun polyglycolide (PGA) scaffold was selected to grow a 3D model of primary bovine endometrial epithelial and stromal cells, that reflects the architecture of the endometrium for the study of pathophysiology. Electrospun scaffolds were seeded with stromal and epithelial cells, and growth was assessed using histological techniques. Prostaglandin E2 and prostaglandin F2α responsiveness of endometrial scaffold constructs was tested using oxytocin plus arachidonic acid (OT + AA) or lipopolysaccharide (LPS). Stromal and epithelial cells growing on the electrospun scaffold had an architectural arrangement that mimicked whole tissue, deposited fibronectin, had appropriate expression of vimentin and cytokeratin and were responsive to OT + AA and LPS, as measured by prostaglandin accumulation. In conclusion, a functional 3D model of stromal and epithelial cells was developed using a PGA electrospun scaffold which may be used to study endometrial pathophysiology.

Loss of WT1 expression in the endometrium of infertile PCOS patients: a hyperandrogenic effect?

CONTEXT: In fertile patients the endometrial Wilms tumor suppressor gene (WT1) is expressed during the window of implantation. Polycystic ovary syndrome (PCOS) patients suffer from hyperandrogenemia and infertility and have elevated endometrial androgen receptor (AR) expression. WT1 is known to be down-regulated by AR. Therefore, the expression of WT1 and its targets may be altered in PCOS endometrium. OBJECTIVE: The objective of the study was to assess the expression and regulation of WT1 and selected downstream targets in secretory endometrium from ovulatory PCOS (ovPCOS) and fertile women. DESIGN AND PATIENTS: Endometrial samples were obtained from 25 ovPCOS and 25 fertile patients. MAIN OUTCOME MEASURE: Endometrial expression of WT1 and selected downstream targets were assessed by immunohistochemistry and RT-PCR. The androgen effect on WT1 expression was determined in vitro by immunoblots and RT-PCR. The expression of WT1 and its targets was quantified in fertile and ovPCOS stromal cells in the presence of androgens by RT-PCR. Caspase-3/7 activity was measured to evaluate sensitivity to drug-induced apoptosis. RESULTS: WT1 expression was down-regulated in secretory-phase ovPCOS endometrium. Stromal expression of Bcl-2 and p27 was higher, and epidermal growth factor receptor was lower in ovPCOS than in fertile patients. Endometrial stromal expression of WT1, Bcl-2, Bcl-2-associated X protein, and ß-catenin was regulated by androgens. Apoptosis levels were reduced in ovPCOS samples and androgen-treated fertile samples. CONCLUSION: WT1 expression is down-regulated in ovPCOS endometrium during the window of implantation. Androgens regulate the expression of WT1 and its targets during endometrial decidualization. The altered balance between WT1 and AR in the endometrium of PCOS patients may jeopardize the success of decidualization and endometrial receptivity.

Interleukin-1ß enhances cartilage-to-cartilage integration.

The failure of cartilages to fuse, particularly in the case of articular cartilage under conditions of repair is due to morphological and structural constraints of the tissue. Factors that impede integration include, non-vascularisation, low cellularity, and proteoglycan in the surrounding extracellular matrix acting as a natural barrier to cellular migration. We hypothesised that brief activation of a catabolic cascade by cytokines followed by culture under anabolic conditions would promote tissue fusion in a ring-disk model of cartilage integration. Our results show that transient exposure to 10 ng mL(-1) interleukin-1ß, followed by two weeks post-culture under anabolic conditions, enhanced cartilage-cartilage integration compared to untreated explants. Quantitative PCR analysis of catabolism-related genes ADAMTS4 and MMP13 showed both were transiently upregulated and these findings correlated with evidence of extracellular matrix remodelling. At the level of histology, we observed chondrocytes readily populated the interfacial matrix between fused explants in interleukin-1ß treated explants, whereas in control explants this region was relatively acellular in comparison. Catabolic cytokine treated explants exhibited 29-fold greater adhesive strength (0.859 MPa versus 0.028 MPa, P 〈 0.05) than untreated counterparts. Collectively, our results demonstrate that a single short catabolic pulse followed by an anabolic response is sufficient to generate mechanically robust, integrative cartilage repair.

Optimized sample preparation for high-resolution AFM characterization of fixed human cells.

Atomic force microscopy enables the simultaneous acquisition of high-resolution topographical and biophysical data allowing integrated analysis of cell surfaces during development and pathogenesis, and, critically, can link molecular and biophysical events. Here we used atomic force microscopy to analyse endometrial epithelial cells and neuronally differentiated P19 cells. Optimized reproducible sample preparation techniques enabled micro- and nanoscale multi-parameter analysis. Comparative analysis using atomic force microscopy and scanning electron microscopy demonstrated the utility of atomic force microscopy for examining tissue morphology, and its ability to generate data allowing differentiation of cells from different origins to be monitored. At low resolution atomic force microscopy produced topographic data complementary to scanning electron microscopy images, whilst at high resolution atomic force microscopy captured novel cell surface structural detail for both epithelial and neuronal cell types. Analysis of surface roughness provided biophysical data which enabled qualitative and quantitative differences between samples to be measured. This study provides an important optimization of sample preparation enabling more generalized atomic force microscopy utilization for cellular analysis required for advanced cell surface morphological studies.

MUC1 as a discriminator between endometrium from fertile and infertile patients with PCOS and endometriosis.

CONTEXT: Endometrium of fertile women expresses progesterone-regulated Mucin 1 (MUC1) that carries selectin ligands recognized by the human blastocyst. Altered MUC1 expression at the time of implantation may contribute to endometrial infertility. OBJECTIVE: The aim was to assess the expression of MUC1 in the endometrium from polycystic ovary syndrome (PCOS), endometriosis, and fertile women in comparison with other hormone-regulated proteins [hydroxysteroid dehydrogenase (HSD) 1, HSD2, estrogen receptor (ER) and progesterone receptor (PR)]. DESIGN AND PATIENTS: Endometrial samples were obtained from 33 fertile patients, 26 ovulatory PCOS patients, 15 anovulatory PCOS patients, and 25 endometriosis patients. MAIN OUTCOME MEASURE: Immunohistochemistry assessed the expression of MUC1 subunits ER, PR, HSD1, and HSD2 in endometrial epithelium. Endometrial MUC1 expression was quantified by immunoblots and RT-PCR. HSD1 and HSD2 expression was assayed by RT-PCR. RESULTS: MUC1ND expression was significantly higher in ovulatory PCOS than in fertile and anovulatory PCOS patients, even after progesterone stimulation. MUC1ND and -CD expression was lower in anovulatory PCOS than in fertile patients. Only MUC1CD expression was lower in endometriosis patients. Endometrial ER expression was significantly higher in PCOS and endometriosis patients, whereas PR expression was significantly higher in PCOS than in fertile patients. The expression of HSD1 was significantly higher in anovulatory PCOS than in fertile patients. Expression of HSD2 was significantly higher in PCOS patients and lower in endometriosis patients. CONCLUSION: Expression of MUC1 subunits in the infertile endometrium is significantly different from fertile and appears to be a component of altered gene expression that potentially contributes to endometrial insufficiency.

L-selectin ligands in human endometrium: comparison of fertile and infertile subjects.

BACKGROUND: L-selectin ligands, localized to the luminal epithelium at the time of implantation, may support the early stages of blastocyst attachment. We have assessed the expression of two L-selectin ligands, defined by MECA-79 and HECA-452 monoclonal antibodies, and the sulfotransferase GlcNAc6ST-2, involved in generation of L-selectin ligand epitopes, in the secretory phase of the endometrium from fertile and infertile patients. METHODS: Endometrial samples were obtained from 33 fertile, 26 PCOS, 25 endometriosis and 33 patients diagnosed with unexplained infertility. L-selectin ligands and GlcNAc6ST-2 expression was assessed by immunohistochemistry and immunoblotting. RESULTS: Immunohistochemical staining of uterine epithelium, from fertile and infertile women, demonstrated differential expression of MECA-79 and HECA-452 epitopes. In fertile women in the secretory phase MECA-79 was more strongly expressed, particularly on the lumen, than in infertile women. HECA-452 staining was significantly stronger in the glands in PCOS and endometriosis patients than in fertile women. GlcNAc6ST-2 expression was reduced in infertile patients, correlating with MECA-79 expression. CONCLUSIONS: This study demonstrated significant differences in expression of L-selectin ligands between fertile and infertile women in natural cycles, and could contribute to patient assessment prior to initiating fertility treatment.

Sfl1 functions via the co-repressor Ssn6-Tup1 and the cAMP-dependent protein kinase Tpk2.

Ssn6 (Cyc8) is a component of the yeast general corepressor Ssn6-Tup1 that inhibits the transcription of many diversely regulated genes. The corepressor does not interact directly with DNA but is recruited to different promoters through interactions with distinct pathway-specific, DNA-binding repressor proteins. Using yeast two-hybrid and GST chromatography interaction experiments, we have determined that Sfl1, a novel repressor protein, interacts directly with Ssn6, and in vivo repression data suggest that Sfl1 inhibits transcription by recruiting Ssn6-Tup1 via a specific domain in the Sfl1 protein. Sin4 and Srb10, components of specific RNA polymerase II sub-complexes that are required for Ssn6-Tup1 repression activity, are found to be required for Sfl1 repression function. These results indicate a possible mechanism for Sfl1-mediated repression via Ssn6-Tup1 and specific subunits of the RNA polymerase II holoenzyme. Electrophoretic mobility shift and chromatin immuno-precipitation assays demonstrate that Sfl1 is present at the promoters of three Ssn6-Tup1-repressible genes; namely, FLO11, HSP26, and SUC2. Sfl1 is known to interact with Tpk2, a cAMP-dependent protein kinase that negatively regulates Sfl1 function. Consistently, we show that phosphorylation by protein kinase A inhibits Sfl1 DNA binding in vitro, and that a tpk2Delta mutation increases the levels of Sfl1 protein associated with specific promoter elements in vivo. These data indicate a possible mechanism for regulating Sfl1-mediated repression through modulation of DNA binding by cAMP-dependent protein kinase-dependent phosphorylation. Taken together with previous data, these new observations suggest a link between cAMP signaling and Ssn6-Tup1-mediated transcriptional repression.

Technical advance: the DNA-binding activity of gal4 is inhibited by methylation of the gal4 binding site in plant chromatin.

Derivatives of the Saccharomyces cerevisiae transcription factor Gal4 which act as effective transcription activators in yeast, Drosophila, mammalian cells and plant protoplasts are shown to direct expression from a GUS reporter construct when expressed in transgenic tobacco. However, in comparison to 35S-GUS controls, Gal4-mediated expression of the reporter gene was relatively weak and extremely variable. GUS expression was lost as plants matured and it was almost undetectable in most of their progeny. Gal4-mediated gene expression could be restored by treating tissues with 5-aza-cytidine, implicating cytosine methylation in the loss of Gal4-mediated expression. Restoration of reporter expression was not accompanied by an increase in steady-state levels of the activator transcript. We propose that the DNA-binding activity of Gal4 is sensitive to methylation of its binding site in plant chromatin. The Gal4-DNA co-crystal predicts that 5-methylcytosine at either of the outer two positions of the binding site will effectively prevent Gal4 binding. We show that these positions become extensively methylated in transgenic plants and that methylation of Gal4-binding sites interferes with Gal4 binding in vitro. These observations suggest that the Gal4 DNA-binding domain is intrinsically sensitive to cytosine methylation and that, despite the success of Gal4-based expression systems in yeast and Drosophila, Gal4 is not ideal for use in plant gene expression technology.

Hrs1/Med3 is a Cyc8-Tup1 corepressor target in the RNA polymerase II holoenzyme.

The Srb/Mediator, a multisubunit subcomplex of the RNA polymerase II (RNA pol II) holoenzyme has been proposed to function as a control panel regulating transcription in response to gene-specific activator proteins. In this report, we identify the Mediator subunit Hrs1/Med3 as a physical target for Cyc8-Tup1, a yeast transcriptional corepressor. Two-hybrid and glutathione S-transferase interaction assays show that Hrs1 can associate directly with Cyc8-Tup1. Moreover, affinity chromatography experiments, using yeast protein extracts, reveal that Cyc8-Tup1 co-purifies with Hrs1 and with additional Mediator subunits in a Hrs1-dependent manner. These observations suggest that Cyc8-Tup1 contacts the Mediator complex via its interaction with the Hrs1 subunit. Further on, genetic analysis indicates that increased Hrs1 dosage can alleviate Cyc8-Tup1-mediated repression, suggesting that Hrs1/Mediator's function is inhibited upon its interaction with Cyc8-Tup1. Finally, artificial holoenzyme recruitment assays support a model by which the contact between the corepressor and the Hrs1/Mediator may prevent pol II holoenzyme recruitment to the core promoter. These data, together with previous genetic evidence, establish a functional and physical interaction between the Cyc8-Tup1 corepressor and the RNA pol II holoenzyme and support a central role of the Mediator complex in transcriptional repression.

The Tup1-Cyc8 protein complex can shift from a transcriptional co-repressor to a transcriptional co-activator.

Cyc8(Ssn6)-Tup1, a general co-repressor complex, is recruited to promoter DNA via interactions with DNA-binding regulatory proteins and inhibits the transcription of many different yeast genes. Previous studies have established that repression function of the complex is performed by one subunit of the complex, the Tup1 protein, and requires specific components of the RNA polymerase II holoenzyme such as Sin4 and Rgr1. In this study we test the transcriptional activity of the Cyc8 subunit using a lexA operator-containing reporter. We show that a LexA-Cyc8 hybrid stimulates transcription when expressed in a tup1Delta, a sin4Delta, or a rgr1Delta strain, suggesting that transcriptional activation is an intrinsic property of the Cyc8-Tup1 co-repressor. In support of this notion we demonstrate that Cyc8-Tup1 has a dual function on CIT2, a gene encoding a citrate synthase that is expressed upon mitochondrial dysfunction. First, we show that Cyc8-Tup1 is tethered to CIT2 promoter by interacting with the activation domain of Rtg3, a bHLH/L-Zip DNA-binding transactivator of CIT2. Next we demonstrate that Cyc8-Tup1 activates CIT2 transcription in response to mitochondrial dysfunction, and this stimulatory effect is mediated by Cyc8. In contrast, basal (noninduced) expression of this gene is inhibited by Tup1. These findings establish a positive role for the Cyc8-Tup1 complex in transcription and support a model by which specific metabolic signals may convert the Cyc8-Tup1 transcriptional co-repressor to a co-activator of certain promoters.

Isolation and characterisation of cDNA clones representing the genes encoding the major tuber storage protein (dioscorin) of yam (Dioscorea cayenensis Lam.).

cDNA clones encoding dioscorins, the major tuber storage proteins (M(r) 32,000) of yam (Dioscorea cayenesis) have been isolated. Two classes of clone (A and B, based on hybrid release translation product sizes and nucleotide sequence differences) which are 84.1% similar in their protein coding regions, were identified. The protein encoded by the open reading frame of the class A cDNA insert is of M(r) 30,015. The difference in observed and calculated molecular mass might be attributed to glycosylation. Nucleotide sequencing and in vitro transcription/translation suggest that the class A dioscorin proteins are synthesised with signal peptides of 18 amino acid residues which are cleaved from the mature peptide. The class A and class B proteins are 69.6% similar with respect to each other, but show no sequence identity with other plant proteins or with the major tuber storage proteins of potato (patatin) or sweet potato (sporamin). Storage protein gene expression was restricted to developing tubers and was not induced by growth conditions known to induce expression of tuber storage protein genes in other plant species. The codon usage of the dioscorin genes suggests that the Dioscoreaceae are more closely related to dicotyledonous than to monocotyledonous plants.