{"paper_id":"fcc82d7b-63ea-494b-96ff-cfab149332aa","body_text":"R E S E A R C H Open Access\nExpression of pluripotency markers in the\nbovine uterus with adenomyosis\nMartyna Łupicka, Barbara Socha, Agata Szczepa ńska and Anna Korzekwa *\nAbstract\nBackground: Adenomyosis is a proliferative uterine dysfunction with unknown aetiology. One possible mechanism of\nits development involves disturbances in stem cell differentiation in uterine tissue. Previously, we identified pluripotent/\nmultipotent cells in the bovine uterus, therefore our present study focused on determining expression of pluripotency\nmarkers, NANOG, OCT4 and SOX2, in bovine adenomyotic tissues and cells.\nFindings: Immunolocalisation revealed protein expression of NANOG, OCT4 and SOX2 in both normal and adenomyotic\nuteri. mRNA expression for NANOG and OCT4 was increased in tissues obtained from uteri with adenomyosis compared\nto controls, but at the protein level there were no significant differences. mRNA expression for all three pluripotency\nmarkers was higher in myometrial cells isolated from uteri with adenomyotic lesions than in those isolated from normal\nuteri. The protein level of NANOG and SOX2 was decreased in stromal cells from adenomyotic tissues, whereas the level\nof OCT4 and SOX2 was increased in myometrial cells obtained from dysfunctional uteri.\nConclusions:The results indicate significant changes in expression of pluripotency markers in adenomyotic compared\nto normal uteri, which suggest the involvement of uterine stem cells in adenomyosis.\nKeywords: Uterus, Pluripotent cells, Adenomyosis, Cow\nBackground\nAdenomyosis is uterine dysfunction characterised by the\npresence of endometrial glands with stromal elements in\nthe myometrium [1]. This pathological condition is well\nrecognized in women, and although it is less known in\ndomestic animals including cows [2 –4], nevertheless it\nmay result in reduced reproductive performance [4].\nAlthough adenomyosis frequently occurs in multiparous\nwomen [5] and cows older than 5 years [4], the aetiology\nof this disorder is still unclear [2]. Several hypotheses\nhave been proposed to explain adenomyosis develop-\nment. One possible mechanism involves the breakdown\nof endometrial and myometrial barrier preceded by\ntrauma such as abortion or gynaecological interventions,\nand followed by reactive hyperplasia of the endometrium\nand its proliferation within the myometrium [1, 6].\nAnother proposed mechanism of adenomyosis develop-\nment involves metaplasia of uterine pluripotent/multipo-\ntent cells under hormonal stimuli. According to this\nhypothesis, glandular nests may arise de novo within the\nmyometrial layer from undifferentiated stem cells under\nspecific conditions, in particular under the influence of\noestradiol (E 2) [7, 8]. Whatever the mechanism under-\nlying formation of glandular foci in the myometrium,\nhormonal and immunological abnormalities certainly\nplay a role during adenomyosis development [9, 10].\nStem cells reside in many adult organs and tissues that\nexhibit high regenerative potential [11]. The cells may be\nidentified by several markers, including NANOG, OCT4\nand SOX2. These proteins are transcription factors\npresent in embryonic stem cells [12] and, as recent stud-\nies have shown, in mesenchymal stem cells settled also\nin reproductive organs [13, 14]. OCT4 and SOX2 are\nprogenitor-specific proteins: octamer-binding transcrip-\ntion factor 4 (OCT4) and sex determine region Ybox 2\n(SOX2). NANOG is a homeodomain-containing tran-\nscription factor and its expression is regulated by\nOCT4/SOX2 heterodimer, which binds to the octamer/\nsox elements at NANOG gene promoter [15]. In the\npresent study we selected NANOG, OCT4 and SOX2 as\nthe markers of undifferentiated state and pluripotency/\nmultipotency of cells that reside in uterus.\n* Correspondence: a.korzekwa@pan.olsztyn.pl\nDepartment of Reproductive Immunology and Pathology, Institute of Animal\nReproduction and Food Research, Polish Academy of Sciences, 10-748\nOlsztyn, Poland\n© 2015 Łupicka et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0\nInternational License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and\nreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to\nthe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver\n(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 \nDOI 10.1186/s12958-015-0106-0\n\nChanges that occur in the endometrium during re-\nproductive cycles, in particular endometrial gland\nmorphogenesis, require a remarkable proliferation\ncapacity of the tissue; thus, pluripotent/multipotent\ncells play an important role in endometrial function-\ning and renewal [11, 16, 17]. These proliferative pro-\ncesses in the uterus remain under the strict control\nof ovarian steroids, therefore these hormones also in-\nfluence uterine stem cell properties [11, 17].\nDuring adenomyosis in cows, protein expression of the\nE2 receptor α (ERα) is increased [4], and also blood and\nendometrial E2 concentrations are elevated, which indicate\nhormonal abnormalities during this condition [4]. Parallel\nwith increased E 2 stimulation, excessive proliferation of\nendometrial cells occurs, which is characterized by expres-\nsion of the proliferation marker KI-67-antigen in adeno-\nmyotic foci [18]. In our recent studies, we identified\npluripotent/multipotent cells in the bovine uterus [19].\nWe also demonstrated expression of the pluripotency\nmarkers NANOG, OCT4 and SOX2 in uterine tissue and\ncultured uterine primary epithelial, stromal and myome-\ntrial cells, and in addition we confirmed pluripotent/mul-\ntipotent properties of these cells by multilineage\ndifferentiation [19]. These results suggest that stem cells\nmay be involved in adenomyosis development in the\nbovine uterus. Therefore, we hypothesized that pluripo-\ntency markers NANOG, OCT4 and SOX2 are differen-\ntially expressed in uterine tissues and cells from control\nand adenomyotic cows. The study by Moreira et al. (2007)\nshowed increased frequency of adenomyosis in cows in\nthe mid luteal stage of the oestrous cycle [20], so for this\nstudy we used uteri from cows at days 8 –10 of the\noestrous cycle.\nThe aims of the study were: (1) comparison of\nNANOG, OCT4 and SOX2 mRNA expression, immu-\nnolocalisation and protein expression in control and\nadenomyotic uterine tissues; (2) determination of\nNANOG, OCT4 and SOX2 mRNA and protein\nexpression in cultured prim ary uterine endometrial\nstromal and myometrial cells isolated from adeno-\nmyotic cows.\nMethods\nMaterial collection\nAll procedures were approved by the Local Animal Care\nand Use Committee, Olsztyn, Poland (agreement no. 83/\n2012/N).\nA total of 24 Holstein/Polish Black and White cows\n(75 %/ 25 %, respectively) 5 –7 years old were used for\npost mortem collection of uteri (days 8 –10 of the\noestrous cycle). Uterine tissues were obtained at the\nMeat Processing Plant “Warmia” (Biskupiec, Poland)\nand transported on ice to the laboratory within 40 min.\nDay of the oestrous cycle was evaluated by macroscopic\nobservation of the ovaries and uterus [21] and confirmed\nby determination of P 4 levels in peripheral blood plasma\nusing radioimmunoassay (RIA). Just before slaughter,\neach animal was examined by a veterinarian via per\nrectum ultrasound-guided examination and information\nabout the age of each cow was recorded. Peripheral\nblood samples were collected from the jugular vein. The\nreasons for culling animals from the herd were eco-\nnomic considerations and herd renewal. For further\nexperiments, after histopathologic examination, material\nquality evaluation and hormone determination, 18 cows\nwere eventually selected (9 for each experimental group).\nTissue fragments (cross-sections of the uterine wall,\ni.e., endometrium and myometrium) were obtained from\nthe middle segment of the uterine horn ipsilateral to the\ncorpus luteum and were divided into three pieces: the\nfirst one was fixed in 4 % paraformaldehyde (PFA) in\n0.1 M PBS (pH 7.4) for histo- and immunohistochemical\nstaining, the second was frozen and stored at −86 °C for\nfurther mRNA and protein expression determination in\nwhole uterine tissue, and the third piece was used for\nimmediate isolation and culture of uterine cells.\nHistochemical staining and preliminary division of the\nmaterial\nUterine tissue was fixed in 4 % PFA and processed for a\nstandard haematoxylin and eosin staining protocol.\nStained cross-sections of the tissue were observed under\na light microscope (Nikon FXA, Tokyo, Japan). Animals\nwere classified as described previously [4]; briefly, if\nuterine glands were present only in the endometrium,\nand if the endometrial-myometrial border was clearly\nvisible, cows were classified as normal/control ( n =9 ,\nFig. 1a). Whereas, if the glands penetrated the myome-\ntrial layer of the uterus, animals were classified as\nadenomyotic ( n = 9, Fig. 1b –d; according to the classifi-\ncation of Katkiewicz et al. 2005 [18]).\nUterine cell isolation and in vitro culture\nEndometrial stromal cells were isolated by enzymatic\ndissociation as previously described [22]. After endomet-\nrial cell isolation, the myometrial layer of the uterus was\naccessed and dissected with scissors. About 4 cm long\nfragments of muscle tissue were chopped up with scis-\nsors into a homogeneous material. Approximately 5 g of\nthe chopped tissue was digested in 50 ml of M199\nmedium (Sigma, M2520, St. Louis, MO, USA) contain-\ning 0.1 % of bovine serum albumin (BSA; Sigma,\nA2058), 20 μg/ml of gentamicin (Sigma, G1271), 2 mg/ml\nof collagenase I (Sigma, C0130), 1 mg/ml of deoxyribo-\nnuclease (Sigma, D5025) and 2 mg/ml of dispase (Life\nTechnologies, 17105–041, Paisley, UK). The enzyme solu-\ntion with myometrial tissue was held at 37.5 °C with stir-\nring for 30 min. After digestion, the cell suspension was\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 Page 2 of 13\n\nfiltered through a mesh to remove undigested tissue frag-\nments, then the cells were washed by centrifugation\n(10 min at 100 x g, at 4 °C). Cells were resuspended in cul-\nture medium (DMEM; Sigma, D5796) supplemented with\n10 % of fetal calf serum (FCS; Sigma, 12133C) and antibi-\notics (gentamicin/amphotericin B; Life Technologies,\n1153727).\nThe cells of each layer of the uterus were seeded sep-\narately at a density of 1 x 10 6 living cells/ml in 1 ml and\n2 ml culture medium per well in collagen-coated 24-well\nand 6-well plates, respectively (Biocoat; BD Bioscience,\n4408, 4400, Bedford, MA, USA), and cultured at 37.5 °C\nin a humidified atmosphere of 5 % CO 2, 95 % air. The\nmedium was changed every 2 days until 70 % confluence\nwas reached (approx. at 4 th day of culture). Total\nmRNA, cell lysates and culture media were collected.\nStromal cells maintained during culture fibroblast-like\nmorphology (Fig. 2a), while myometrial cells exhibited\nfusiform appearance (Fig. 2b). Purity of the cell cultures\nwas rated by 4 independent observations under the light\nmicroscope based on cells morphology [23], and was\nevaluated for approx. 90 –95 % for each cell types. Cell\nculture homogeneity was also confirmed using real-time\nPCR for determination of mRNA expression of vimentin\nand desmin for stromal and myometrial cells, respect-\nively [23, 24]. Vimentin was highly expressed in stromal\ncells and in contrary weakly expressed in myometrial cell\ncultures (Fig. 2c). Desmin was expressed mainly in myo-\nmetrial cultures, while in stromal cultures the expression\nwas on low level (Fig. 2d). For cells ’ functionality\nconfirmation prostaglandin (PG) E 2 and PGF 2α level was\nmeasured in culture medium by enzyme immunoassay\n(EIA; Fig. 3a, b). The levels of secreted PGs indicate\nmaintained functionality of the uterine cells during cul-\ntures [25 –27].\nImmunohistochemistry\nImmunohistochemistry (IHC) was used to localise\nnuclear transcription factors, NANOG, OCT4 and\nSOX2, in uterine tissues.\nCross-sections of uterine horn samples were fixed in\n4 % PFA in 0.1 M PBS (pH 7.4), and cryoprotected in\n18 % sucrose. Immunostaining was carried out on con-\nsecutive 7 μm cryostat sections. To block endogenous\nperoxidase, the sections were treated with hydrogen per-\noxide in methanol and washed in 0.1 M PBS. The sec-\ntions were blocked with 10 % normal goat serum\n(Sigma, G9023) for 1 h at room temperature (approx.\n23 °C; RT), incubated overnight at RT with a 1:100 dilu-\ntion of anti-NANOG (Abcam, 80892, Cambridge, UK),\nanti-OCT4 (Abcam, 19857) or anti-SOX2 (Sigma,\nS9072) antibodies, washed in PBS, incubated for 1 h\nwith a 1:25,000 dilution of biotinylated anti-rabbit\n(Vectastain ABC Kit; Vector Laboratories, PK 4001,\nBurlingame, CA, USA) antibodies, then washed, incu-\nbated for 45 min with the ABD reagent in PBS, and\nwashed again. Proteins were visualized by incubating\nthe sections in 0.3 mg/mL 3,30-diaminobenzidine tet-\nrahydrochloride in 0.01 % hydrogen peroxide in Tris-\nbuffered saline (pH 7.2) for 2 –3 min. Finally, the\nFig. 1 Representative pictures of haematoxylin and eosin stained bovine uterine cross-section slides. a – normal uterine tissue with regular, clearly\nvisible border between endometrium and myometrium; b-d – adenomyotic tissue with visible uterine glands within the myometrial layer of the\nuterus. Arrows indicate glandular nests in myometrium. Scale bars: 100 μm\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 Page 3 of 13\n\nsections were dehydrated and cover-slipped with DPX\nmounting medium (Park Scientific Ltd, D-11601,\nNorthampton, UK). To determine the specificity of\nthe immunohistochemical staining, two controls were\nperformed: first, the primary antibody was omitted\nduring the immunostaining procedure; second, the\nprimary antibody was substituted with a nonspecific\nIgG. Observations and photographs were made with a\nlight microscope (Nikon FXA).\nTotal RNA isolation\nTotal RNA was extracted from uterine tissues (approx.\n30 mg) and cultured cells using TRI-Reagent (Sigma,\nT9424) according to the manufacturer ’s instructions.\nThe content and purity of RNA was assessed on a\nNanoDrop 1000 (Thermo Fisher Scientific, ND-1000,\nWilmington, DE, USA). 260/280 absorbance ratio for\nall samples was approx. 2.0, and 260/230 absorbance\nratio ranged between 1.8 –2.2. One microgram of each\n0\n1\n2\n3\n*\nvimentin/GAPDH mRNA expression\n(arbitrary units)\nstromal myometrial\n0\n2\n4\n6\n8\n*\ncells\ndesmin/GAPDH mRNA expression\n(arbitrary units)\nC\nAB\nD\nFig. 2 Evaluation of cell cultures homogeneity. a, b – representative pictures of stromal and myometrial cultured cells, respectively. Scale bars: 20μm.\nExpression of vimentin in stromal and myometrial cells c; expression of desmin in stromal and myometrial cells d. Data were normalized\nagainst glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars represent the mean ± SEM. Asterisks indicate statistical difference between uterine\nstromal and myometrial cells (P < 0.05), as determined by Student’s t-test\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 Page 4 of 13\n\nsample of total RNA was reverse-transcribed to cDNA\nwith the QuantiTect Reverse Transcription kit (Life\nTechnologies, 205313), as described in the supplier ’s\nprotocol. The cDNA obtained was stored at −20 °C\nuntil real-time PCR was applied.\nReal-time PCR quantification\nmRNA expression for NANOG, OCT4 and SOX2 in the\ntissues and cells was determined by quantitative real-\ntime PCR. The experiments were performed using the\nApplied Biosystems 7900 (Applied Biosystems, Foster\nCity, CA, USA) with SensiFAST SYBR Hi-ROX Kit (Bio-\nline Reagents, BIO-92002, London, UK) according to the\nmanufacturer’s instructions. The real-time PCR reaction\nmix (20 μl) contained 19 μl of SensiFAST SYBR Hi-ROX\nMaster Mix, 0.5 μM of sense and antisense primers, and\n1 μl of reverse-transcribed cDNA (50 ng). Primer se-\nquences used for determination of vimentin, desmin,\nNANOG, OCT4, SOX2 and glyceraldehyde 3-phosphate\ndehydrogenase ( GAPDH) mRNA expression are detailed\nin Table 1. Standard curves consisting of serial dilutions\nof the appropriate cDNA were plotted for efficiency\nevaluation. Amplification was preceded by an initial en-\nzyme activation step (2 min, 95 °C). The PCR steps were\nas follows: 40 cycles of denaturation (5 s, 95 °C), then\nannealing and extending (20 s, 60 °C). After amplifica-\ntion, melting curves were acquired by stepwise increases\nat a temperature of 50 –95 °C to ensure that a single\nproduct was amplified and no primer-dimer structures\nwere formed. Control reactions in the absence of the\ntemplate or primers were performed to confirm that\nproducts were free from primer-dimers and genomic\nDNA contamination, respectively. Dissociation curves\nanalysis was carried out after each real-time experiment\nto confirm the presence of only one amplification prod-\nuct. Data were normalized using the ΔΔ Ct method.\nSamples were amplified in duplicates. Data are shown as\nthe average fold increase, with S.E.M., and are expressed\nrelative to the housekeeping gene GAPDH.\nWestern blotting\nProtein expression for NANOG, OCT4 and SOX2 in the\ntissues and cells was determined by Western blotting.\nProteins from homogenised tissues and in vitro cultured\ncells were released by incubationwith lysis buffer containing\n50 mM Tris –HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA,\n0.1 % SDS, 1 % TritonX-100, 0.5 % sodium deoxycholate\nand protease inhibitors (Sigma, P8340). The lysates were\nstored at −86 °C until further analysis. Protein concentra-\ntions were measured by the Bradford’sm e t h o d .\nWestern blot analysis was performed as previously\ndescribed [28]. Equal amounts of protein were dis-\nsolved in SDS gel-loading buffer, heated to 95 °C for\n4 min and separated in 10 % SDS-PAGE. Separated\nproteins were electroblotted onto 0.2 μm nitrocellu-\nlose membranes in transfer buffer. After blocking in\n5 % non-fat dry milk in TBS-T buffer for 1.5 h at RT, the\nmembranes were incubated overnight with a 1:250 dilution\nof anti-NANOG (Novus Biologicals Ltd, NBP2-24941,\nCambridge, UK), a 1:400 dilution of anti-OCT4 (Novus\nBiologicals Ltd, NB100-2379) or a 1:500 dilution of anti-\nSOX2 (Sigma, S9072) antibodies; GAPDH (Sigma, G8795;\nmonoclonal anti-glyceraldehyde-3-phosphate dehydrogen-\nase antibody produced in mouse) expression was used as a\nreference. Proteins were detected by incubating the mem-\nbranes with a 1:20,000 dilution of secondary polyclonal\nanti-rabbit or anti-mouse alkaline phosphatase-conjugated\nantibodies (Sigma, A 3687, A 3562) for 1.5 h at RT.\nWestern blots were quantitated using the Kodak 1 D\nprogram (Eastman Kodak, Rochester, NY, USA).\nHormone determination\nMeasurements of P 4 in blood plasma were performed\nusing a direct radioimmunoassay (RIA; DIASource\n0\n1000\n2000\n3000\n4000\nPGE2 (pg/ml)\nstromal myometrial\n0\n500\n1000\n1500\n2000\n2500\ncells\nPGF2  (pg/ml)\nA\nB\nFig. 3 Prostaglandins secretion by stromal and myometrial cultured\ncells. Secretion of prostaglandin E2 by cultured uterine cellsa;s e c r e t i o n\nof prostaglandin F 2α by cultured uterine cells b.B a r sr e p r e s e n t\nthe mean ± SEM. There were no statistically significant differences\nin PGs secretion between stromal and myometrial cells ( P > 0.05),\nas determined by Student ’s t-test\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 Page 5 of 13\n\nImmunoAssays S.A., KIP1458, Nivelles, Belgium).\nThe standard curve ranged from 0.12 –36 ng/ml and\nthe effective dose for 50 % inhibition (ED 50) of the\nassay was 0.05 ng/ml. The intra- and inter-assay coeffi-\ncients of variation (CV) were 6.5and 8.6 %, respectively.\nProstaglandins determination\nMeasurements of PGE 2 and PGF 2α levels in culture\nmedia were performed using commercially available en-\nzyme immunoassay kit (EIA kit; Cayman Chemical\nCompany, 514010 for PGE 2 and 516011 for PGF 2α, Ann\nArbor, MI, USA). Standard curve for PGE 2 ranged from\n9,5–5000 pg/ml and the effective dose for 50 % inhib-\nition (ED 50) of the assay was 15 pg/ml. The intra- and\ninter-assay coefficients of variation (CV) were 4.2 % and\n12.4 %, respectively. PGF 2α standard curve ranged from\n9,5–2000 pg/ml, ED 50 of the assay was 9 pg/ml and the\nintra- and inter-assay CV were on average 9.4 % and\n12 %, respectively.\nStatistical analysis\nStatistically significant differences between groups in the\nexperiments were evaluated using Student ’s t-test\n(GraphPad PRISM Version 5.00, San Diego, CA, USA).\nAll data were expressed as means ± SEM. Differences\nwere analysed between control and adenomyotic cows,\nand were considered significant at P < 0.05.\nResults\nmRNA expression, immunolocalisation and protein\nexpression of pluripotency markers NANOG, OCT4 and SOX2\nin uterine tissue of cows with adenomyosis\nmRNA expression of transcription factors NANOG\nand OCT4 was increased in adenomyotic uterine tissue\ncompared with normal uteri ( P < 0.05, Fig. 4a, b).\nThere was no significant difference in SOX2 mRNA\nexpression between control and adenomyotic uteri\n(P > 0.05, Fig. 4c).\nImmunohistochemistry revealed expression of all three\npluripotency markers, NANOG, OCT4 and SOX2, in nor-\nmal uterine tissues (Fig. 5c, e, g) as well as in adenomyotic\nsamples (Fig. 5d, f, h). The proteins examined were mainly\nlocalised in the endometrium, however, in the case of ade-\nnomyotic tissues the immunoreactivity was also high in the\nmyometrial compartment of the uterus and in adenomyo-\ntic, ectopic glands (Fig. 5d, f, h).\nAt the protein expression level, determined by Western\nblotting, there were no significant differences among\nNANOG, OCT4 and SOX2 ( P > 0.05, Fig. 6a–c). However\nthe spatial differences in examined proteins expression was\nreported during IHC assay.\nmRNA and protein expression of pluripotency markers\nNANOG, OCT4 and SOX2 in uterine cells from cows with\nadenomyosis\nExpression of all genes of the three transcription factors\ndid not differ significantly between cultured stromal cells\nisolated from adenomyotic uteri compared to those iso-\nlated from control tissues ( P > 0.05, Fig. 7a –c). Whereas,\nmRNA expression of NANOG, OCT4 and SOX2 in cul-\ntured uterine myometrial cells isolated from cows with\nadenomyosis was increased compared to those isolated\nfrom normal uteri ( P < 0.05, Fig. 7d –f) .\nProtein expression of NANOG and SOX2 was signifi-\ncantly decreased in stromal cells isolated from uteri with\nadenomyosis compared to those obtained from normal\nuteri ( P < 0.05, Fig. 8a, c). Protein expression of both\ntranscription factors OCT4 and SOX2 was higher in cul-\ntured myometrial cells from adenomyotic tissues than in\nTable 1 Oligonucleotide sequences used for real-time PCR\nGene Oligonucleotide sequences Product size GeneBank\n(bp)\nvimentin FWD 5 ’-GACCTGGAGCGTAAAGTGG-3’ 108 BC118269\nREV 5’-GACATGCTGTTCTTGAATCTGG-3\ndesmin FWD 5 ’-GACCCAGGCAGCCAACAAG-3’ 100 BC133410\nREV 5’-GTCGATCTCGCAGGTGTAGG-3’\nNANOG FWD 5 ’-TGCATTTGCTGGAGACTGAG-3’ 107 DQ069776\nREV 5’- GTCCCGGTCAAGAAACAAAA-3’\nOCT4 FWD 5 ’-AGGTGTTCAGCCAAACGACTA-3’ 195 FD381287.1\nREV 5’-TCTCCTGCAGATTCTCGTTGT-3’\nSOX2 FWD 5 ’-GCACATGAACGGCTGGAGCAACG-3’ 218 JQ231229.1\nREV 5’-TGCTGCGAGTAGGACATGCTGTAGG-3’\nGAPDH FWD 5 ’-CACCCTCAAGATTGTCAGCA-3’ 103 BC102589\nREV 5’-GGTCATAAGTCCCTCCACGA-3’\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 Page 6 of 13\n\ncorresponding cells isolated from normal uteri ( P < 0.05,\nFig. 8e, f ).\nDiscussion\nAdenomyosis is a uterine proliferative dysfunction which\naetiology is still unclear. One possible mechanism of its\ndevelopment implies the involvement of uterine stem\ncells, which abnormal proliferation and differentiation\nmay lead to formation of glandular foci within the myo-\nmetrium [2, 12]. In our previous study, we confirmed\nthe existence of pluripotent/multipotent cells in the\nbovine uterus [19]. The present research focused on\ndetermining expression of pluripotency markers in ade-\nnomyotic uterine tissues and cells. We confirmed that\npluripotency markers are expressed in adenomyotic\nendometrial tissues and in glandular nests within the\nmyometrium, and that mRNA expression for NANOG\nand OCT4 was higher in dysfunctional tissue compared\nto the control; however, the tissue protein results deter-\nmined by Western blotting did not confirm these differ-\nences. Moreover, we have demonstrated that both\nmRNA and protein levels for OCT4 and SOX2 were in-\ncreased in cultured primary myometrial cells isolated\nfrom adenomyotic uteri compared to the cells isolated\nfrom normal tissues. However, in stromal cells protein\nexpression of NANOG and SOX2 was significantly de-\ncreased in the case of adenomyosis. To our knowledge,\nthis is the first study to report expression of pluripotency\nmarkers in the bovine uterus with adenomyosis.\nIn women, the hypothesis of uterine stem cell involve-\nment in development of uterine dysfunction has been\nwidely studied [11, 29 –31]. Moreover, the recent report\nof Chen et al. (2014) showed increased expression of\nanother stem cell marker, Musashi-1, in adenomyotic\neutopic and ectopic endometrium of women [32]. The\npresence of pluripotent/multipotent cells in the bovine\nuterus suggests their possible role in development of\nuterine dysfunction [19, 33, 34]. Nevertheless, there are\nno studies on the involvement of pluripotent cells in\npathogenesis of adenomyosis or any other uterine dis-\neases in cows. Our present study is consistent with one\nconducted in women [32], because we demonstrated in-\ncreased expression of pluripotency markers in myome-\ntrial cells isolated from adenomyotic uteri. However, in\n0\n1\n2\n3\n4\n*\nOCT4/GAPDH mRNA expression\n(arbitrary units)\nC ADENO\n0\n1\n2\n3\n4\nSOX2/GAPDH mRNA expression\n(arbitrary units)\n0.0\n0.5\n1.0\n1.5\n2.0\n2.5\n*\nNANOG/GAPDH mRNA expression\n(arbitrary units)\nA\nB\nC\nFig. 4 mRNA expression of pluripotency markersNANOG a, OCT4 b and\nSOX2 c in uterine tissues obtained from control cows and from cows\nwith adenomyosis. Data were normalized against glyceraldehyde-3-\nphosphate dehydrogenase (GAPDH). Bars represent the mean ± SEM.\nAsterisks indicate statistical difference between uterine normal and\nadenomyotic tissues (P < 0.05), as determined by Student’s t-test. C –\ntissues obtained from control cows, ADENO– tissues obtained from\ncows with adenomyosis\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 Page 7 of 13\n\nFig. 5 Immunodetection of pluripotency markers in uterine tissues from control cows and from cows with adenomyosis. a, b – haematoxylin and\neosin stained slides of control and adenomyotic uterus, respectively;c, d – NANOG immunodetection in control and adenomyotic tissue, respectively;\ne, f – immunolocalisation of OCT4 in normal and adenomyotic tissue, respectively;g, h – SOX2 immunodetection in control and adenomyotic tissue,\nrespectively;i, j, k – no Ab, negative controls for NANOG, OCT4 and SOX2, respectively. Unspecific IgG controls (pictures notshown) served similar pictures\nas no Ab control. Arrows indicate the most intense histochemical reactions; dotted line indicate endometrial-myometrial border; e– endometrium, m–\nmyometrium, es– endometrial stroma, eg– endometrial gland, a– adenomyotic lesion, v– vessel. Scale bars: 20μm\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 Page 8 of 13\n\nwhole uterine tissue, at the protein level evaluated by\nWestern blotting we did not find significant differences\nin NANOG, OCT4 and SOX2 expression, in contrast to\nour mRNA results. Moreover, mRNA expression of plur-\nipotency markers in stromal primary cells also did not\nreflect protein expression. The reason for this inconsist-\nency may be posttranslational modifications of the pro-\nteins, which results in different protein expression\npattern when comparing to mRNA. However in myome-\ntrial cells mRNA expression was consistent with protein\nexpression. These outcomes together with IHC results\nindicate that the pattern of pluripotency markers expres-\nsion in adenomyotic tissue may depends on the uterine\ncompartment: endometrium or myometrium. Our ex-\nperiments performed on the tissue showed general\nexpression of pluripotency markers in adenomyotic\nuterus, which included its expression in stem cells that\nmigrate to uterus through blood and lymphatic vessels,\ne.g. cells of medullary origin [31]. Whereas in vitro ex-\nperiment revealed pluripotency markers expression in\nparticular uterine cells, originated from endometrium or\nmyometrium. In our previous study we showed that the\nmain source of stem cells in the bovine uterus is stromal\nlayer [19], therefore we showed changed expression\npattern of pluripotency markers in case of uterine\npathology – adenomyosis.\nIn our study, we also demonstrated decreased protein\nexpression of NANOG and SOX2 in cultured primary\nendometrial stromal cells from adenomyotic tissues\ncompared to the controls, which suggests that their\npotential to differentiate into glands within the endomet-\nrium may be reduced. Proliferation of endometrial cells\nand formation of uterine glands is extremely important\nfor successful implantation and early embryo development\n[35]. In high fertility heifers, endometrial expression of\ngenes involved in cell proliferation, tissue morphology\nand development was increased when compared to\nlow fertility heifers [36]. Thus, during adenomyosis,\ndisturbed proliferative processes in the endometrium\nof cows may impair their fertility [18, 37]. Moreover,\nbecause the myometrium is a prolific source of pluripo-\ntent cells during adenomyosis, this may imply a higher\ndifferentiation potential of cells in this compartment\n0.0\n0.5\n1.0\n1.5\n2.0\n2.5\nOCT4/GAPDH protein expression\n(arbitrary units)\nC ADENO\n0.0\n0.5\n1.0\n1.5\n2.0\nSOX2/GAPDH protein expression\n(arbitrary units)\n0.0\n0.5\n1.0\n1.5\nNANOG/GAPDH protein expression\n(arbitrary units)\nA\nB\nC\nD\nFig. 6 Protein expression of NANOG a,O C T 4 b and SOX2 c in\nbovine uterine tissues obtained from control cows and from\ncows with adenomyosis. Data were normalized against glyceraldehyde-\n3-phosphate dehydrogenase (GAPDH). Bars represent the mean ± SEM.\nThere were no statistical differences between uterine normal and\nadenomyotic tissues ( P > 0.05), as determined by Student ’s t-test.\nRepresentative blots for NANOG, OCT4, SOX2 and GAPDH are\nshown below the graphs d.M M – molecular weight marker, C –\ntissues obtained from control cows, ADENO – tissues obtained\nfrom cows with adenomyosis\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 Page 9 of 13\n\nof the uterus, and may trigger the invasion of glandular\nnests deep within.\nMaintenance of uterine cell functions, including prolif-\neration, during cyclic endometrial remodelling is the\nmain factor underlying female fertility in many species\nand is controlled by ovarian hormones [29, 37 –39]. In\nwomen, abnormalities in endometrial cell proliferation\npotentially leads to development of gynaecological\ndiseases such as endometriosis, endometrial cancers and\nadenomyosis [11, 29]. Bovine endometrial cell prolifera-\ntion is also regulated by ovarian hormones: oestradiol\nand progesterone [40]. Adenomyosis is an oestrogen-\ndependent dysfunction [4, 30], thus it is suggested that\nhormone disturbances may relate to uterine stem cell\nfunctioning during adenomyosis [29]; however, this issue\nrequire further studies. In mares, defective responses of\n0.0\n0.5\n1.0\n1.5\nNANOG/GAPDH mRNA expression\n(arbitrary units)\n0.0\n0.5\n1.0\n1.5\n2.0 *\nNANOG/GAPDH mRNA expression\n(arbitrary units)\n0.0\n0.5\n1.0\n1.5\n2.0\nOCT4/GAPDH mRNA expression\n(arbitrary units)\n0.0\n0.5\n1.0\n1.5\n2.0\n*\nOCT4/GAPDH mRNA expression\n(arbitrary units)\nC ADENO\n0\n1\n2\n3\n4\nstromal cells\nSOX2/GAPDH mRNA expression\n(arbitrary units)\nC ADENO\n0\n1\n2\n3\n4\n**\nmyometrial cells\nSOX2/GAPDH mRNA expression\n(arbitrary units)\nA\nB\nC\nD\nE\nF\nFig. 7 mRNA expression of pluripotency markers in uterine cells isolated from control cows and from cows with adenomyosis. NANOG a OCT4\nb and SOX2 c mRNA expression in uterine stromal cells. NANOG d OCT4 e and SOX2 f mRNA expression in uterine myometrial cells. Data were\nnormalized against glyceraldehyde-3-phosphate dehydrogenase ( GAPDH). Bars represent the mean ± SEM. Asterisks indicate statistical differences\nbetween uterine normal and adenomyotic tissue (* P < 0.05; **P < 0.01), as determined by Student ’s t-test. C – cells obtained from control cows,\nADENO – cells obtained from cows with adenomyosis\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 Page 10 of 13\n\n0.0\n0.5\n1.0\n1.5\n2.0\n**\nNANOG/GAPDH protein expression\n(arbitrary units)\n0.0\n0.5\n1.0\n1.5\nNANOG/GAPDH protein expression\n(arbitrary units)\n0.0\n0.5\n1.0\n1.5\n2.0\n2.5\nOCT3/4/GAPDH protein expression\n(arbitrary units)\n0.0\n0.5\n1.0\n1.5\n2.0\n*\nOCT3/4/GAPDH protein expression\n(arbitrary units)\nC ADENO\n0.0\n0.5\n1.0\n1.5\n*\nstromal cells\nSOX2/GAPDH protein expression\n(arbitrary units)\nC ADENO\n0.0\n0.5\n1.0\n1.5\n*\nmyometrial cells\nSOX2/GAPDH protein expression\n(arbitrary units)\nA\nB\nC\nD\nE\nF\nG\nFig. 8 (See legend on next page.)\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 Page 11 of 13\n\nendometrial glands to cyclic hormonal stimuli may con-\ntribute to degenerative changes in the endometrium,\ntermed endometrosis, and result in decreased fertility\n[41]. These changes are also linked to functional abnor-\nmalities of endometrial cells, especially impaired prolif-\neration activity in endometrotic glandular nests [39].\nAlthough knowledge about the involvement of uterine\nstem cells in pathogenesis of endometrosis is poor, re-\ncent studies on mare infertility caused by this dysfunc-\ntion suggest the utility of stem cell transplantation into\nuteri for therapy. Stem cells that settled in degenerative\nendometria during the experiments of Mambelli et al.\n(2014) induced proliferation of glands and improved\ntheir secretory functions [42]. These data suggest that a\nwide range of uterine pathologies in different species\nmay be dependent on functions of uterine stem cells.\nTherefore, our present study contributes by broadening\nknowledge about this issue in cattle, which was not pre-\nviously studied in this species.\nConclusions\nIn conclusion, our results indicate that significant changes\noccur in the expression of pluripotency markers,\nNANOG, OCT4 and SOX2, in bovine adenomyotic tis-\nsues compared to normal uteri. Moreover, this expression\nwas dependent on the uterine compartment: in general, it\nwas decreased in the endometrial layer and the converse\nin the myometrial layer. These data suggest the involve-\nment of uterine multipotent/pluripotent cells in develop-\nment of adenomyosis. Therefore our study imply that\nstem cells play role not only under physiological condi-\ntions but also in the case of uterine pathologies in cows.\nCompeting interests\nThe authors declare that they have no competing interests.\nAuthors’ contributions\nMŁ and AK designed the study. M Ł performed all experiments, acquired and\nanalysed the data. AS and BS participated in experiments execution and\nmaterial collection. M Ł and AK drafted the manuscript. M Ł revised the\nmanuscript. All authors read and approved the final manuscript.\nAcknowledgements\nThe authors wish to thank W. Krzywiec for technical support and tissue\ncollection and M. Domin - the owner of the slaughterhouse (Meat\nProcessing Plant “Warmia”, Biskupiec, Poland) for permitting collection of the\nmaterial.\nThis work was supported by Grant-in-Aid for Scientific Research from the\nNational Science Centre (2011/01/B/NZ5/00939).\nReceived: 18 June 2015 Accepted: 17 September 2015\nReferences\n1. Azziz R. 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Changes in expression pattern of selected endometrial proteins\nfollowing mesenchymal stem cells infusion in mares with endometrosis.\nPlos One. 2014;9:e97889.\nSubmit your next manuscript to BioMed Central\nand take full advantage of: \n• Convenient online submission\n• Thorough peer review\n• No space constraints or color figure charges\n• Immediate publication on acceptance\n• Inclusion in PubMed, CAS, Scopus and Google Scholar\n• Research which is freely available for redistribution\nSubmit your manuscript at \nwww.biomedcentral.com/submit\nŁupicka et al. Reproductive Biology and Endocrinology  (2015) 13:110 Page 13 of 13","source_license":"CC0","license_restricted":false}