A Novel Mouse Model of Endometriosis Mimics Human Phenotype and Reveals Insights into the Inflammatory Contribution of Shed Endometrium

• DOI: 10.1016/j.ajpath.2014.03.011
• Publication Date: 2014-06-05T01:15:34Z
• Authors: Erin Greaves; Fiona L. Cousins; Alison Murray; Arantza Esnal-Zuf...; Amelie Fassbender; Andrew W. Horne; Philippa T.K. Sau...
• Keywords: Peritoneal cavity

Endometriosis is an estrogen-dependent inflammatory disorder characterized by the presence of endometrial tissue outside uterine cavity. Patients experience chronic pelvic pain and infertility, with most likely origin deposits (lesions) being fragments shed at menses. Menstruation process associated a dramatic increase in mediators tissue-resident immune cells. In present study, we developed validated mouse model endometriosis using syngeneic menstrual introduced into peritoneum immunocompetent mice. We demonstrate establishment endometriotic lesions that exhibit similarities to those recovered from patients undergoing laparoscopy. Specifically, both cases, had epithelial (cytokeratin+) stromal (vimentin/CD10+) cell compartments well-developed vasculature (CD31+ endothelial cells). Expression estrogen receptor β was increased compared or eutopic endometrium. By performing experiments mice green fluorescent protein–labeled macrophages (MacGreen) reciprocal transfers wild-type mice, obtained evidence menses endometrium can contribute microenvironment lesions. summary, exhibits human peritoneal respect expression, inflammation, macrophage infiltration, providing opportunity for further studies possible identification novel therapies this perplexing disorder. occurs 6% 10% women reproductive age,1Giudice L.C. Kao Endometriosis.Lancet. 2004; 364: 1789-1799Abstract Full Text PDF PubMed Scopus (2593) Google Scholar represents huge socioeconomic burden.2Simoens S. Dunselman G. Dirksen C. Hummelshoj L. Bokor A. Brandes I. Brodszky V. Canis M. Colombo G.L. DeLeire T. Falcone Graham B. Halis Horne Kanj O. Kjer J.J. Kristensen J. Lebovic D. Mueller Vigano P. 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Endocrine regulation menstruation.Endocr Rev. 2006; 17-46Crossref (410) where luminal portion sheds end cycle. primary cause widely accepted be introduction cavity via fallopian tubes, known as retrograde menstruation.7Sampson J.A. Metastatic embolic endometriosis, due dissemination venous circulation.Am J Pathol. 1927; 3: 93-110.43PubMed captive baboons, incidence spontaneous parallels number cycles experienced, link between shedding onset disease.8D'Hooghe T.M. Bambra C.S. De Jonge Lauweryns J.M. Koninckx P.R. prevalence baboon (Papio anubis, Papio cynocephalus) increases duration captivity.Acta Obstet Gynecol Scand. 1996; 75: 98-101Crossref (45) Current treatment strategies are restricted surgical excision suppression function mimic premature menopause. up 75% symptoms recur after surgery, long-term sometimes ineffective.9Giudice Clinical practice: endometriosis.N Engl Med. 2010; 362: 2389-2398Crossref (1346) Although it acknowledged there unmet clinical need nonsurgical treatments their development depends access animal models recapitulate features disorder.10Rogers P.A. Fazleabas Giudice Montgomery G.W. Petraglia F. Taylor R.N. Defining future directions research: workshop report 2011 World Congress Montpellier, France.Reprod Sci. 20: 483-499Crossref (115) Nonhuman primates offer physiologically relevant although use limited cost ethical concerns. Injecting female baboons autologous has been shown result lesions.11D'Hooghe Kyama C.M. Chai Fassbender Vodolazkaia Mwenda primate translational research endometriosis.Reprod 16: 152-161Crossref (98) This used longitudinal studies, allowing insights changes gene they become established.12Afshar Y. Hastings Roqueiro Jeong J.W. A.T. Changes during progression experimental baboon, anubis.Biol 88: 44Crossref (58) same model, aromatase isoform (ERβ) found lesions, finding agreement women.13Bukulmez Hardy D.B. Carr B.R. Word R.A. Mendelson C.R. Inflammatory status influences steroid endometriosis.Endocrinology. 2008; 149: 1190-1204Crossref (153) 14Fazleabas Brudney Langoi Steroid lesions.Fertil Steril. 2003; 80: 820-827Abstract (112) Rat have also developed, offering advantage lower smaller size testing potential therapies. availability genetically engineered express marker proteins15Sasmono R.T. Oceandy Pollard Tong W. Pavli Wainwright B.J. Ostrowski M.C. Himes S.R. Hume D.A. A colony-stimulating factor receptor-green protein transgene expressed throughout mononuclear phagocyte system mouse.Blood. 101: 1155-1163Crossref (500) 16Wilkosz Pullen N. de-Giorgio-Miller Ireland Herrick Cellular exchange endometriosis-adhesion GFP transgenic mice.Gynecol Invest. 2011; 72: 90-97Crossref (9) targeted deletion genes implicated disease progression17Fang Yang Lydon J.P. DeMayo Tamura Gurates Intact progesterone receptors essential counteract proliferative effect estradiol endometriosis.Fertil 82: 673-678Abstract (44) made them attractive alternative studies. To date, usually involved injection immunosuppressed mice18Hull M.L. Johan M.Z. Hodge W.L. Robertson S.A. Ingman W.V. Host-derived TGFB1 deficiency suppresses lesion endometriosis.Am 180: 880-887Abstract (63) small sutured other sites (reviewed Grummer19Grummer Animal research.Hum 12: 641-649Crossref (158) Scholar). Results provided its association infertility20Grummer Translational study endometriosis-associated infertility.Semin 31: 125-132Crossref (18) served platform therapies, including targeting angiogenesis lesions21Hull Charnock-Jones D.S. Chan C.L. Bruner-Tran K.L. Osteen K.G. Tom B.D. Fan T.P. Smith S.K. Antiangiogenic agents effective inhibitors 2889-2899Crossref (228) Scholar; however, limitations. For example, injected either under skin formation endometriosis-like these grow if recipients lack capacity mount rejection response, eg, homozygous PrkdcSCID mutation.18Hull Models intact allow role(s) cytokines cells,22Lin Isacson Axonal growth embryonic stem cell-derived dopaminergic neurons Netrin-1 Slits.Stem Cells. 24: 2504-2513Crossref (60) 23Somigliana Zingrillo Ranieri Filardo Candiani Vignali Induction inhibits spleen leukocyte function.Acta 2001; 200-205Crossref (4) but because do not decidualization menstruation, does time menstruation. Furthermore, autologous/syngeneic models, secured local abrasion and/or sutures,16Wilkosz 22Lin procedures induce artificial response. set out develop would combine best current models. our recently menstruation24Cousins F.L. Murray Esnal Gibson Evidence migration mesenchymal-epithelial transition rapid restoration integrity menstruation.PLoS One. 2014; 9: e86378Crossref (77) source determine whether approach could estrogenic examined complemented undertaking which granulocyte/macrophage lineage were labeled (GFP).15Sasmono Mature (approximately 8-week-old) C57BL/6 purchased Harlan Laboratories (Derby, UK) allowed acclimate 1 week before surgery. Some performed Cfs1r-EGFP originally generated described detail elsewhere.15Sasmono founder stocks gift Bernadette Dutia Prof. David (The Roslin Institute, University Edinburgh). Two heterozygous enhanced GFP+ males two females cross-bred (WT) form breeding colony bred standard conditions. Offspring genotyped classified MacGreen WT. Mice maintained chow water available ad libitum housed environmentally controlled facilities illuminated 7:00 am pm. All accordance UK legal requirements licensed approval Home Office (London). induced adult donor 8 weeks age) protocol in-house24Cousins (Figure 1A). brief, ovariectomized (day 1) primed s.c. injections 100 ng estradiol-17β (days 7 9), (P4; Sigma-Aldrich, Dorset, delivered SILASTIC implant (Dow Corning Corp, Midland, MI) days 13 19, 5 sesame oil 13, 14, 15. Decidualization one horn 20 μL 15), decidualized culled day 4 hours P4 withdrawal (removal pellet) opening longitudinally petri dish scraping away myometrial layer scalpel 1, B C). mass suspended 0.2 mL PBS passed through 19-gauge needle i.p. anesthetized recipient previously implanted (Sigma-Aldrich) Tissue inoculate each 40 mg tissue/0.2 per mouse). Three injection, (photographs body taken carefully dissected surrounding tissue) tissues fixed 4% normal buffered formaldehyde histologic analysis placed RNA Save (Geneflow Ltd, Lichfield, extraction. total, 18 Of these, 10 transfers: WT (n = 6) 4). Peritoneal biopsy samples 19) (aged 45 years) laparoscopic surgery pain. Endometrial 18) explorative laparoscopy no detected (controls). collected informed consent provided, Lothian Research Ethics Committee. Eighty-two recruited, subsequently taking exogenous hormones 3 months sample collection used, 37 analyzed study. Cycle stage determined serum levels staining based Noyes criteria,25Noyes Hertig Rock Dating biopsy.Fertil 1950; 1: 3-25Abstract secretory phases analyzed. Further details Table 1. Biopsy RNAlater reagent (Applied Biosystems, Warrington, quantitative RT-PCR neutral-buffered formalin immunohistochemical (IHC) analysis.Table 1Patient CharacterizationCharacteristicEndometriosisNo endometriosisSample (No.)1918Age [years (means ± SD)]33.68 6.90332.38 9.193Proliferative (No.)88Secretory (No.)1110 Open table tab After fixation, stained H&E; containing identifiable analysis. Single-color IHC according protocols24Cousins 26Collins MacPherson Bombail Williams A.R. Anderson Jabbour oestrogen receptors, ERα, ERβ, ERβ variants, cancers prostaglandin F may play regulating ERα.BMC Cancer. 330Crossref (66) citrate antigen retrieval followed blocking endogenous peroxidase 3% H2O2 methanol. streptavidin-biotin block (Vector Laboratories, Peterborough, performed, any nonspecific reduced species-specific solution (1:4 Tris-buffered saline + 5% bovine albumin). Incubation appropriate dilution antibody overnight 4°C (Table 2). Biotinylated secondary antibodies (dilution 1:500) diluted incubated room temperature 30 minutes. streptavidin–horseradish conjugate 1:1000; Sigma-Aldrich) hour, visualization ImmPACT diaminobenzidine substrate Laboratories). anti-CD31 analysis, epitopes blocked Bloxall (Dako Ely, UK), streptavidin–alkaline phosphatase visualized PermaRed Ltd). Images captured AxioVision software version 4.8.2.0 (Carl Zeiss Cambridge, Provis microscope (Olympus America Inc., Center Valley, PA). Dual immunofluorescence herein (ab) polyclonal IgG (horseradish peroxidase), detection TSA kit (PerkinElmer, Waltham, MA) Cy3 (red) fluorescein (green). second antigen, sections microwaved 2.5 minutes boiling buffer, applied 4°C. Secondary system, counterstained DAPI 1:500). Slides mounted PermaFluor medium (Thermo Fisher Scientific, MA). Sections evaluated LSM 710 confocal ZEN 2009 Ltd).Table 2List Antibodies Used AnalysisAntibody specificity (supplier)Raised inUsed onDilutionERβ [AbD Serotec (#MCA1974S)]MouseHuman1:50ERβ [Santa Cruz Biotechnology (#SC-8974)]RabbitMouse1:500ERα Laboratories)MouseHuman mouse1:50CD31 (Dako)RabbitMouse1:800Cytokeratin [Sigma-Aldrich (#C2562)]MouseMouse1:2000Vimentin [Cell Signaling Technology Inc. (#5741)]RabbitMouse1:600GFP [Invitrogen Molecular Probes (#A11122)]RabbitMouse1:250Biotinylated anti-mouse Laboratories)Goat1:500Biotinylated anti-rabbit Laboratories)Goat1:500Horseradish peroxidase–conjugated streptavidin (Dako)NA1:200NA, applicable. NA, Total extracted homogenization TRI Reagent (Life Technologies Paisley, Qiagen TissueLyser 25 Hz minutes, chloroform phase separation lysates processed RNeasy (Qiagen Valencia, CA). Complementary DNA synthesized SuperScript VILO (Invitrogen, starting template concentration approximately RNA. Real-time PCR reactions Roche Universal ProbeLibrary (Roche Applied Science, West Sussex, supermix (Invitrogen). Quantitative 7900 fast real-time machine Technologies, Grand Island, NY), 18S control. Primer sequences supplied 3.Table 3List SequencesSpeciesmRNAForwardReverseHumanERα5′-TTACTGACCAACCTGGCAGA-3′5′-ATCATGGAGGGTCAAATCCA-3′HumanERβ5′-ATCATGGAGGGTCAAATCCA-3′5′-TGGGCATTCAGCATCTCC-3′MouseERα5′-GCTCCTAACTTGCTCCTGGAC-3′5′-CAGCAACATGTCAAAGATCTCC-3′MouseERβ5′-CCTCAGAAGACCCTCACTGG-3′5′-CACGCACTTCCCCTCATC-3′MouseIL-65′-GCTACCAAACTGGATATAATCAGGA-3′5′-CCAGGTAGCTATGGTACTCCAGAA-3′MouseCCL25′-CATCCACGTGTTGGCTCA-3′5′-GATCATCTTGCTGGTGAATGAGT-3′MouseCCL55′-TGCAGAGGACTCTGAGACAGC-3′5′-GAGTGGTGTCCGAGCCATA-3′MouseTNFα5′-CTGTAGCCCACGTCGTAGC-3′5′-TTGAGATCCATGCCGTTG-3′ Statistical D'Agostino Pearson omnibus normality test one-way variance Newman-Keuls post-test Student's unpaired t-test. Graphpad Prism (GraphPad Software San Diego, CA) perform statistical tests. inherent differences counterpart (it shed), confirmed comparable histological corresponding cycle stages (Supplemental Figure S1). estrogen-dominated estrous stage, glands uterus round S1A), S1C). diestrous, larger tortuous S1B), S1D). induction withdrawal, culled, 1B), 1C). material, identified anterior posterior walls parietal 1D); adipose bladder visceral covering uterus, gut, intestines; mesentery gut kidney; underneath kidneys. observations patients, exhibited macroscopic microscopic heterogeneity. Phenotypes observed included brown/black examination, demonstrated hemosiderin hemorrhage 2A). Lesions neutral color 1D) surrounded 2B), fluid-filled cysts 2E). similar patients: fibrotic 2C), 2D), cystic appearance 2G). 2, A, B, E, F) D H) glandular epithelium. high proportion (83.3%) 4); mean 2.06 mouse, 81% contained Not all material adhered peritoneum, floating harvest, consistent reports researchers.27Burns K.A. Rodriguez K.F. Hewitt S.C. Janardhan K.S. Young S.L. Korach Role signaling required model.Endocrinology. 153: 3960-3971Crossref ScholarTable 4Information about Recovered Mouse Model PatientsVariableMouseHumanSample (No.)1820Recovery rate (%)83.3100Number (mean SD)2.06 0.32≥1Lesions stroma (%)8168 Immunostaining identify phenotype (anti-cytokeratin) 3B), fibroblasts (anti-CD10) 3D), (anti-CD31) 3F), identical types 3, C, E). significantly more vascularized SEM: 133.1 19.62 44.55 26.54, respectively; P < 0.05) 3G) (511.6 159.1 116 25.7, 3H). reported patterns human28Critchley Brenner R.M. Henderson T.A. K. Nayak N.R. Slayden O.D. Millar M.R. β, α, vascular endothelium nonhuman endometrium.J 86: 1370-1378Crossref (208) mouse29McNeilly J.R. Taggart Cranfield Cooke H.J. McNeilly A.S. Loss oocytes Dazl knockout results steroidogenic altered gonadotropin secretion animals.Endocrinology. 2000; 141: 4284-4294Crossref Concentrations mRNAs 4A) 4B) (P 0.001 0.01, respectively). contrast, concentrations mRNA homogenates higher than 0.05 both). Additional data patient comparing given Supplemental S2; significant difference wa