Study design
The main objective of this study was to evaluate the role of MIG-6 in endometrial P4 resistance. First, the expression of MIG-6 was assessed in eutopic endometrium of infertile women with endometriosis compared to fertile women. To determine whether endometriosis affects MIG-6 expression, we examined MIG-6 expression in a nonhuman primate and mouse model of endometriosis. Subsequently, we identified ERBB2 as a MIG-6 target and evaluate the impact of Erbb2 ablation on the infertility and endometrial P4 resistance of Mig-6d/d mice. Finally, transcriptomic analysis was applied to dissect the molecular mechanisms of Mig-6 in the uterus. The control and treatment groups and the number of biological replicates (sample sizes) for each experiment are specified in the figure legends. Animal numbers for each study type were determined by the investigators on the basis of previous experience with the standard disease models that were used or from pilot studies. Animals were randomly allocated to the control and treatment groups and housed together to minimize environmental differences and experimental bias. Analysis of endpoint readouts was carried out in a blinded fashion.
Ethics Statement
The institutional review board of Michigan State University, Greenville Health System, and University of North Carolina approved this study. The Institutional Animal Care and Use Committee at Michigan State University approved all experiments relating to mice. The Institutional Animal Care and Use Committees of both the University of Illinois at Chicago and Michigan State University approved the endometriosis baboon animal model.
Human Endometrium Samples
The human endometrial samples used to examine MIG-6 expression patterns were obtained from Michigan State University’s Center for Women’s Health Research Female Reproductive Tract Biorepository, the University of North Carolina, and the Greenville Hospital System in accordance with the guidelines set by the Institutional Review Boards of Michigan State University (Grand Rapids, MI), the University of North Carolina (Chapel Hill, NC), and Greenville Health System (Greenville, SC), respectively. Written informed consent was obtained from all participants. For experiments examining MIG-6 mRNA expression throughout the menstrual cycle, endometrial samples were analyzed from 22 cycling premenopausal women without endometriosis (n = 6 proliferative, n = 7 early secretory, n = 3 mid secretory, and n = 6 late secretory) and from 20 cycling premenopausal women with endometriosis (n = 2 proliferative, n = 6 early secretory, n = 9 mid secretory, and n = 3 late secretory). Control endometrial tissues were laparoscopically negative for endometriosis and had not been on any hormonal therapies for at least three months prior to surgery. Endometrial menstrual staging was confirmed by an experienced pathologist familiar with female reproduction. To investigate MIG-6 amounts in the endometrium from women, 10 control and 10 eutopic endometrium with endometriosis were used. To compare MIG-6 amounts in the eutopic endometrium and ectopic lesions of women with endometriosis, each of 12 samples were used. All women with endometriosis were infertile. Samples used for immunohistochemistry were fixed in 10% buffered formalin prior to embedding in paraffin wax.
Animals And Tissue Collection
Animals were maintained in a designated animal care facility according to Michigan State University’s Institutional Guidelines for the care and use of laboratory animals. All animal procedures were approved by the Institutional Animal Care and Use Committee of Michigan State University. For all animal studies, animals were randomly distributed among different conditions by the investigator as the animals did not show any size or appearance differences at the onset of the experiments. No animals were excluded, and the investigator was not blinded to group allocation during the experiment. Erbb2 conditional knockout mice were generated by crossing Pgrcre/+Mig-6f/f with Erbb2f/f mice (Pgrcre/+Mig-6f/fErbb2f/f; Mig-6d/dErbb2d/d). Pregnant uterine samples were obtained by mating control (Mig-6f/f or Mig-6f/fErbb2f/f), Mig-6d/d and Mig-6d/dErbb2d/d female mice with C57BL/6 male mice the morning of a vaginal plug designated as day 0.5 of gestation (GD 0.5). Mice were sacrificed at GD 3.5 and 5.5. For the study of steroid hormone regulation, control, Mig-6d/d and Mig-6d/dErbb2d/d mice at 6 weeks of age were ovariectomized. Two weeks postsurgery, ovariectomized mice were injected with vehicle (sesame oil; Veh) or estradiol (0.1 µg/mouse; E2) plus progesterone (1 mg/mouse; P4) for 3 days and euthanized at 6 hours after injection. For the fertility studies, adult female control, Mig-6d/d and Mig-6d/dErbb2d/d mice were placed with wild type C57BL/6 male mice. The mating cages were maintained for 6 months and the number of litters and pups born during that period was recorded. Uterine tissues were then immediately processed at the time of dissection and either fixed with 4% (vol/vol) paraformaldehyde for histology or immunohistochemistry or snap frozen and stored at -80 °C for RNA/protein extraction.
Induction Of Endometriosis
For baboon uterine samples, endometriosis was induced by intraperitoneal inoculation of menstrual endometrium on two consecutive menstrual cycles and harvested using laparotomy via endometriectomy from four female baboons as previously described 55. For mouse uterine samples, 8-weeks-old female mice which have conditional double-fluorescent Cre reporter gene (Pgrcre/+Rosa26mTmG, Pgrcre/+ Mig-6f/fRosa26mTmG, and Pgrcre/+ Mig-6f/fErbb2f/f Rosa26mTmG were injected with 1 µg/ml of E2 per a day at three times and had a surgical procedure to induce endometriosis. Under anesthesia, a midline abdominal incision was made to expose the uterus in female mice, and one of uterine horn was removed. In a Petri dish containing phosphate-buffered saline (PBS; pH 7.5), the uterine horn was opened longitudinally with scissors. The excised uterine horn was cut into small fragments of about 1 mm3, and then injected back into the peritoneum of same mouse. The abdominal incision and wound were closed with sutures and skin was closed with surgical wound clips, respectively. After a designated time, the mice were euthanized, and endometriosis-like lesions were removed using a fluorescence microscope and counted.
Endometriosis-related Infertility Analysis
Endometriosis were induced in 8 week old control female mouse recipient (fertile) receiving endometrial fragments from donor control (Pgrcre/+Rosa26mTmG) or Mig-6d/d Rosa26mTmG endometrium. A sham surgery group was included as a control. After 1, 2, and 3 months of the endometriosis induction, the mice with endometriotic lesions of control or Mig-6d/d were mated with wild-type male mice and then collected at GD 7.5.
RNA Isolation And Microarray Analysis
Total RNA was extracted from the uterine tissues using the RNeasy Total RNA Isolation Kit (Qiagen, Valencia, CA). RNA was pooled from the uteri of more than three mice per genotype at GD 3.5 and microarray analysis was performed using GeneChip® Mouse Genome 430 2.0 Arrays (Affymetrix) as described previously 56(Gene Expression Omnibus accession code GSE138185). Array data were analyzed using Bioconductor for quantile normalization. We selected aberrantly expressed genes in the uteri of control, Mig-6d/d and Mig-6d/dErbb2d/d mice at GD 3.5 using a two-sample comparison according to significant fold change greater than 1.5. Aberrantly expressed genes were classified with canonical pathway analyzed by Ingenuity System Software (Ingenuity Systems Inc.).
Reverse Transcription - Quantitative PCR
The complementary DNAs (cDNAs) were synthesized with MMLV Reverse Transcriptase (Invitrogen Crop) according to the manufacturer’s instructions. RT-qPCR was performed on cDNA to assess the expression of genes of interest with SYBR Green (Bio-Rad) or TaqMan primers (Applied Biosystems). Experimental gene expression data were normalized against the housekeeping gene, 18S ribosomal RNA. Analysis of mRNA expression was first undertaken by the standard curve method, and results were corroborated by cycle threshold values assessing gene expression. Primer sequences used in these studies are shown in table S4.
Immunohistochemistry Analyses
Immunohistochemistry and immunofluorescence analyses were performed as previously described 57. Briefly, dewaxed hydrated paraffin-embedded tissue sections were pre-incubated with 10% normal goat (for anti-MIG-6, Ki67, Cyclin D1, ErbB2, pERK1/2, ERK1/2, MUC1, LTF, and KLF4 antibodies) or donkey (for anti-MCM2, MCM6, and KLF15 antibodies) serum in PBS and then incubated with anti-MIG-6 (1:200 dilution; Customized antibody by Dr. Jeong Lab), anti-Ki67 (1:1000 dilution; #ab15580; Abcam), anti-Cyclin D1 (1:1000 dilution; #eo-RB9041-p0; Thermo Fisher Scientific), anti-ErbB2 (1:200 dilution; #2165; Cell Signaling), anti-pERK1/2 (1:500 dilution; #4370; Cell Signaling), anti-ERK1/2 (1:1000 dilution; #4695; Cell Signaling), anti-MUC1 (1:1000 dilution; #ab15481, Abcam), anti-LTF (1:2000 dilution; #07-682, Millipore Corp.), anti-MCM2 (1:20000 dilution; #sc9839, Santa Cruz Biotechnology), anti-MCM6 (1:20000 dilution; #sc9843; Santa Cruz Biotechnology), anti-KLF4 (1:5000 dilution; #sc20691; Santa Cruz Biotechnology), and anti-KLF15 (1:5000 dilution; #ab2647; Abcam) antibodies in PBS supplemented with 10% normal serum overnight at 4 °C. For immunohistochemistry, the sections were incubated with secondary antibody conjugated to horseradish peroxidase (Vector Laboratories) for one hour at room temperature. Immunoreactivity was detected using diaminobenzidine (DAB-Vector Laboratories) and analyzed using microscopy software from NIS Elements, Inc. (Nikon). A semi-quantitative grading system (H-score) was calculated to compare the immunohistochemical staining intensities. The H-score was calculated using the following equation: H-score = ∑ Pi (i), where i = intensity of staining with a value of 1, 2, or 3 (weak, moderate, or strong, respectively) and Pi is the percentage of stained cells for each intensity, varying from 0 to 100%. The overall score ranged from 0 to 300 58.
Western Blot Analysis
Western blot analyses were performed as described previously 59. Proteins were extracted using lysis buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2.5 mM EDTA, and 0.125% Nonidet P-40 (vol/vol)) supplemented with both a protease inhibitor cocktail (Roche, Indianapolis, IN) and a phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein lysates were electrophoresed via SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). Membrane was blocked with Casein (0.5% w/v) in PBS with 0.1% Tween 20 (v/v; Sigma-Aldrich) prior to exposure to anti-ErbB2 (#2165; Cell Signaling, Danvers, MA), anti-EGFR (#2646; Cell Signaling), anti-phospho-ERK1/2 (pERK1/2; #4370; Cell Signaling), anti-ERK1/2 (#4695; Cell Signaling), anti-MIG-6 (Customized antibody by Dr Jeong Lab) or anti-β-actin (#sc1616; Santa Cruz Biotechnology) antibodies diluted to 1:1000. Immunoreactivity was visualized by incubation with a horseradish peroxidase-linked secondary antibody followed by exposure to Electrochemiluminescence reagents (ECL) according to manufacturer’s instructions (GE Healthcare Biosciences).
Statistical Analysis
No statistical method was used to predetermine sample size for in vivo studies. Based on prior experience, all experiments used 5 mice per group to achieve adequate statistical power. For all animal experiments, block randomization was used to ensure a balance in sample size across groups. The investigators were blinded during the evaluation of results variations in the group. For all animal experiments, over three biological replicates were analyzed for each condition, and results are presented as the mean ± SEM. For data with only two groups, Student’s t test was used. For data containing more than two groups, an analysis of variance (ANOVA) test was used, followed by Tukey test for pairwise t-test. p < 0.05 was considered statistically significant. All statistical analyses were performed using the Instat package from GraphPad. The original data are provided in table S5.
Data Availability
All data are available in the manuscript or the supplementary material. The accession number for microarray generated in this study is GSE138185.