Fig 1.
Immunocompetent mouse model of endometriosis.
6–8 weeks-old CD1 female mice were primed with PMSG for 48 hours. Uterine tissues were then harvested and minced into tiny cell aggregates after myometrial removal. Female mice with the same genetic background were subjected to ovariectomy and served as recipients. 2 weeks after surgery, equal volumes of uterine cell aggregate suspension were transferred into the peritoneal cavities of recipients. Endometriosis was maintained by subcutaneous administration of 100 ng of E2 once every 4 days until tissue collection. For the studies of the role of P4 in endometriosis, 1 mg of P4 (pre-P4) was administrated along with E2 beginning at 4 days before transplantation. For P4-resistance experiments (Post-P4), 1 mg of P4 was administration along with E2 beginning at 4 days after transplantation. The ectopic lesions were assessed under a dissecting microscope at different days after endometrial cell transplantation (n = 6 per treatment group).
Fig 2.
P4 alleviates E2-dependent establishment and growth of ectopic lesions.
Ectopic lesions were established in the peritoneal cavities of immunocompetent female mice and treated with E2 or P4 along with E2 as described in Fig 1 (N = 6). (A) Locations of ectopic lesions (Dashed circles). (B) Histology of ectopic lesions. Dashed lines indicate the contact sites of the ectopic lesions with adjacent peritoneum. Representative images of ectopic lesions harvested at four weeks after induction are shown (40X). (C) Time-course progression of ectopic lesions. The average volumes of the ectopic lesions at the indicated time points are shown. (D) The average numbers of ectopic lesions identified in each treatment group are shown. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as #: p < 0.05, *: p<0.01.
Fig 3.
P4 inhibits E2-dependent cell proliferation and angiogenesis in ectopic lesions.
Sections of the ectopic lesions collected from E2 or E2 plus P4-treated recipients (D16, n = 6) were subjected to histological examination. (A) Representative images (20X) showing H&E and Trichrome staining, or IHC analysis using antibody against myofibroblast biomarker αSMA, uterine epithelial biomarker KRT11, or uterine stromal biomarker VIM, respectively. (B) Representative images (20X) showing IHC analysis using antibodies against cell proliferation biomarker KI67, smooth muscle biomarker αSMA, endothelial cells CD31, or an angiogenetic regulator CCN1, respectively. The numbers of KI67-positive cells, the perimeters of the supporting blood vessels, and the immunostaining intensities of CCN1 and CD31 were analyzed by ImageJ software. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as #: p < 0.05, *: p<0.01.
Fig 4.
P4 suppresses E2-dependent inflammatory responses in the ectopic lesions.
Ectopic lesions were harvested from E2 or E2 plus P4-treated recipients (n = 6) by 16 days after induction. (A) Representative images (20X) showing IHC analysis using antibodies against pan-macrophage biomarker (F4/80), inflammatory M1 (CCR7), anti-inflammatory M2 (CD206) macrophages or Treg cell biomarker (FOXP3), respectively. (B) The relative level of mRNA expression corresponding to Ccl2, Ccl5, Il1b, Il6, Tnfa, and Tgfb was analyzed by qPCR after moralization to the internal control gene, 36B4. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as #: p < 0.05, *: p<0.01.
Fig 5.
Loss of ERα/PR-mediated signaling contributes to P4-resistance in this mouse model of endometriosis.
Endometriosis was induced and maintained with E2 as described in Fig 1. The host females were then treated with P4 beginning at 4 days before (Pre-) or 4 days after (Post-) endometrial cell transplantation until tissue collection (n = 6). Donor uterine tissue (D0) and ectopic lesions were subjected to IHC analysis (A) for ERα, PR and HAND2 protein expression (20X) or qPCR analysis (B) to assess expression level of mRNA corresponding to Esr1, Pgr, Hand2, and Hoxa10, respectively. (C) Lesion volumes were quantitated by 16 days after induction. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as #: p < 0.05, *: p<0.01.
Fig 6.
DNA methylation is involved in regulation of PR-signaling in endometriosis.
Endometriosis was induced in female mice and maintained with E2. Beginning at 4 days after induction, host females were treated with either vehicle (VEH) or 0.5 mg/kg of Decitabine (DAC) intraperitoneally, once every other day until tissues collection (n = 6). (A) Lesion volumes were measured at 8 and 16 days after induction. (B) The eutopic endometrium (EU) and the ectopic lesions (EC) were subjected to IHC analysis for PGR protein expression (20X). Representative images from each group are shown. The relative intensities of PR staining were analyzed by ImageJ software. (C) qPCR was employed to analyze relative levels of gene expression in the ectopic lesions (D16) treated with VEH or DAC. (D) DNA methylation levels of Hoxa10 and Pgr promoters were assessed by qPCR following digestion of genomic DNA with methylation-specific enzymes. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as #: p < 0.05, *: p<0.01.