Role of estrogen receptor signaling required for endometriosis-like lesion establishment in a mouse model

Abstract

Endometriosis results from ectopic invasion of endometrial tissue within the peritoneal cavity. Aberrant levels of the estrogen receptor (ER), ERα and ERβ, and higher incidence of autoimmune disorders are observed in women with endometriosis. An immunocompetent mouse model of endometriosis was used in which minced uterine tissue from a donor was dispersed into the peritoneal cavity of a recipient. Wild-type (WT), ERα-knockout (αERKO), and βERKO mice were donors or recipients to investigate the roles of ERα, ERβ, and estradiol-mediated signaling on endometriosis-like disease. Mice were treated with vehicle or estradiol, and resulting location, number, and size of endometriosis-like lesions were assessed. In comparison with WT lesions in WT hosts, αERKO lesions in WT hosts were smaller and fewer in number. The effect of ER status and estradiol treatment on nuclear receptor status, proliferation, organization, and inflammation within lesions were examined. αERKO lesions in WT hosts did not form distal to the incision site, respond to estradiol, or proliferate but did have increased inflammation. WT lesions in αERKO hosts did respond to estradiol, proliferate, and show decreased inflammation with treatment, but surprisingly, progesterone receptor expression and localization remained unchanged. Only minor differences were observed between WT lesions in βERKO hosts and βERKO lesions in WT hosts, demonstrating the estradiol-mediated signaling responses are predominately through ERα. In sum, these results suggest ER in both endometriosis-like lesions and their environment influence lesion characteristics, and understanding these interactions may play a critical role in elucidating this enigmatic disease.

Fig. 1.

Endometriosis experimental protocol. WT, αERKO, and βERKO host mice were ovariectomized (OVEX) on d −14 and were given a sc injection of corn oil or estradiol valerate (2.5 μg/mouse·wk) once a week for the duration of the study beginning at OVEX (*). WT, αERKO, and βERKO donor mice were primed with pregnant mare serum gonadotropin (PMSG) (10 IU) 41 h before removal of uterus for uterine synchronization. Donor uteri were cleanly removed, washed thrice in PBS, slit longitudinally, and finely minced. Minced uterine tissue was injected into the peritoneal cavity of a host mouse (n = 6–12 per group). Endometriosis-like lesions were removed 21 d after minced uterine tissue injection.

Fig. 2.

Evaluation of endometriosis-like lesion formation using WT, αERKO, and βERKO immunocompetent mice as donors or recipients. A, Estradiol signaling mediated through ERα confers a maximal number of endometriosis-like lesions. Adherent lesions were removed from WT to WT, WT to αERKO, WT to βERKO, αERKO to WT, and βERKO to WT lesions from mice treated with vehicle or estradiol (E2) (2.5 μg/mouse·wk) for 3 wk and counted. Letters indicate P < 0.05 by one-way ANOVA with Tukey's multiple-comparison posttest. B, Endometriosis-like lesions require ERα to increase in weight with estradiol treatment. *, P < 0.05, unpaired one-tailed t test. C, Representative macroscopic and microscopic photomicrographs of endometriosis-like lesions. Macroscopic images are at ×7.5 or ×14.5 magnification, and microscopic images stained with H&E are ×100 or ×400 magnification. Yellow arrows indicate endometriosis-like lesions; white arrows in 1 and 19 indicate uterus; white arrows in 24 indicate intestine.

Fig. 3.

An increase in the proliferative marker Ki67 is seen after estradiol (E2) treatment in endometriosis-like lesions. Representative photomicrographs demonstrating Ki67 expression in WT to WT, WT to αERKO, WT to βERKO, αERKO to WT, and βERKO to WT lesions from mice treated with vehicle or estradiol (2.5 μg/mouse·wk) for 3 wk. Magnification, ×400.

Fig. 4.

PR localization does not redistribute from the epithelial to the stromal compartment with estradiol (E2) treatment in the WT to αERKO group. Representative photomicrograph demonstrating PR expression in WT to WT, WT to αERKO, WT to βERKO, αERKO to WT, and βERKO to WT lesions from mice treated with vehicle or estradiol (2.5 μg/mouse·wk) for 3 wk. Magnification, ×200.

Fig. 5.

PR gene expression does not increase with estradiol treatment in WT to αERKO lesions, and all lesions except the αERKO to WT lesions are responsive to estradiol (E2) treatment when examining estradiol-responsive genes Ltf and Muc4. A, ERα and PR gene expression. B, Ltf, Muc4, and Krt18 gene expression. Total RNA was isolated from WT to WT, WT to αERKO, WT to βERKO, αERKO to WT, and βERKO to WT endometriosis-like lesions. Mice were treated with vehicle (corn oil) or estradiol (2.5 μg/mouse·wk) for 3 wk. Transcripts were quantified by real-time PCR as described under Materials and Methods and were normalized relative to RpL7. Letters indicate P < 0.05 by one-way ANOVA with Bonferroni's posttest; *, P < 0.05, unpaired one-tailed t test.

Fig. 6.

Endometriosis-like lesions from WT to αERKO and αERKO to WT vehicle-treated mice have higher inflammation scores, with the WT to αERKO increased inflammation score resolved with estradiol (E2) treatment. A, Inflammation score of endometirosis-like lesions. Lesions fixed for histology were stained by H&E and immunohistochemically for CD3 and F4/80 to assess degree of inflammation. When the inflammatory cells involved less than 10, 11–30, 31–60, and more than 60% of the tissue section, the inflammation was graded as minimal (1), mild (2), moderate (3), and severe (4), respectively. B, Gene expression analysis of inflammatory markers. Total RNA was isolated from WT to WT, WT to αERKO, WT to βERKO, αERKO to WT, and βERKO to WT endometriosis-like lesions. Mice were treated with vehicle (corn oil) or estradiol (2.5 μg/mouse·wk) for 3 wk. Transcripts were quantified by real-time PCR as described under Materials and Methods and were normalized relative to RpL7. Letters indicate P < 0.05 by one way ANOVA with Tukey's multiple-comparison posttest.

Fig. 7.

Key angiogenic markers are not increased with estradiol (E2) treatment in αERKO uteri. Total uterine RNA was isolated from WT and αERKO mice treated with vehicle (0.85% saline/0.25% ethanol) or estradiol (250 ng/mouse) for 2 h. Transcripts were quantified by real-time PCR as described under Materials and Methods and were normalized relative to RpL7. *, P < 0.05, unpaired one-tailed t test.

Fig. 8.

Estrogen signaling through ERα is required for endometriosis-like lesion growth and responsivity. A schematic representation is shown of the roles of ERα, ERβ, and estradiol in endometriosis-like disease pathology in an immunocompetent mouse model. Symbols denote where ERα, ERβ, or estradiol are important for endometriosis-like lesions.