Endometrial Expression of Steroidogenic Factor 1 Promotes Cystic Glandular Morphogenesis

Abstract

Epigenetic silencing of steroidogenic factor 1 (SF1) is lost in endometriosis, potentially contributing to de novo local steroidogenesis favoring inflammation and growth of ectopic endometrial tissue. In this study, we examine the impact of SF1 expression in the eutopic uterus by a novel mouse model that conditionally expresses SF1 in endometrium. In vivo SF1 expression promoted the development of enlarged endometrial glands and attenuated estrogen and progesterone responsiveness. Endometriosis induction by autotransplantation of uterine tissue to the mesenteric membrane resulted in the increase in size of ectopic lesions from SF1-expressing mice. By integrating the SF1-dependent transcriptome with the whole genome binding profile of SF1, we identified uterine-specific SF1-regulated genes involved in Wingless and Progesterone receptor-Hedgehog-Chicken ovalbumin upstream promoter transcription factor II signaling for gland development and epithelium-stroma interaction, respectively. The present results indicate that SF1 directly contributes to the abnormal uterine gland morphogenesis, an inhibition of steroid hormone signaling and activation of an immune response, in addition to previously postulated estrogen production.

Figure 1.

Enhanced glandular development. A, Immunohistochemical staining for SF1, MYC-tag, and FOXA2 in representative uterine cross-sections of 8- to 10-week-old ovariectomized females. B, Quantification of glands and glandular size. C, RT-qPCR analysis for Foxa2 and Dcc message. D, RT-qPCR analysis for Wnt4 mRNA levels. E, Immunostaining of β-catenin. S, stroma; GE, glandular epithelium; LE, luminal epithelium. Two tailed t test significance indicated by ***, P < .001; **, P < .01; *, P < .05 and NS, not significant. SF1^LSL/+ n = 8 and Pgr^cre/+ SF1^LSL/+ n = 6.

Figure 2.

An aging uterine phenotype. A, Summary of litter production in a 6-month breeding period. B, Gross morphology of uterine horns in 8-month-old cycling females. Scale bar, 1 cm. C, Uterine to body weight ratio. Significance indicated by *, P < .05. D, Eosin and hematoxylin staining of uterine cross-sections. Lower panels are high magnification of the boxed area in top panel. Arrows indicate endometrial compartments: S, stroma; GE, glandular epithelium; LE, luminal epithelium. Two tailed t test significance indicated by *, P < .05. SF1^LSL/+ n = 5 and Pgr^cre/+ SF1^LSL/+ n = 5.

Figure 3.

SF1 in endometriosis. A, Immunohistochemical staining of SF1 in human eutopic endometrium and ovarian endometriomas. S, stroma; E, epithelium. B, Gross morphology of ectopic lesions in a murine endometriosis model. C, Quantification of ectopic lesion size. D, Immunohistochemical staining for BrdU incorporation in proliferating cells of ectopic lesions. E, Quantification of epithelial proliferation. Two tailed t test significance indicated by *, P < .05. SF1^LSL/+ n = 9 and Pgr^cre/+ SF1^LSL/+ n = 7.

Figure 4.

KEGG pathways enriched in genes differentially regulated by SF1 expression in murine endometrium. A, Top KEGG pathway terms. B, RT-qPCR validation of gene expression in uteri of 8- to 10-week-old ovariectomized females. Two tailed t test significance indicated by *, P < .05; **, P < .01; ***, P < .001. SF1^LSL/+ n = 8 and Pgr^cre/+ SF1^LSL/+ n = 6.

Figure 5.

Global genomic binding of SF1 in the endometrium determined by ChIP-Seq. A, Distribution of SF1-binding intervals around transcriptional start site of annotated genes in uteri of 8- to 10-week-old ovariectomized females. B, Enrichment of SF1-binding intervals relative to expected genomic distribution. C, SeqPos motif enrichments analysis of SF1-binding intervals identified the top enriched motifs with representative motif depicted by a graph of the position-specific scoring matrix.

Figure 6.

Validation of direct SF1 targets. A, Venn diagram comparison of genes bound by SF1 within 10 kb of gene boundaries and genes with differential expression by microarray in uteri of 8- to 10-week-old ovariectomized females. B, ChIP-qPCR validation of direct targets in independent uterine samples from 8- to 10-week-old ovariectomized females. C, Schematic for genes encoding enzymes for synthesis of E₂ from the cholesterol precursor. D, RT-qPCR validation of expression of steroidogenic genes in independent uterine samples from 8- to 10-week-old ovariectomized females. Two tailed t test significance indicated by *, P < .05 and ***, P < .001. SF1^LSL/+ n = 8 and Pgr^cre/+ SF1^LSL/+ n = 6.

Figure 7.

A model for the role of SF1-dependent regulatory network for uterine gland formation. SF1 promotes gland formation by increasing Wnt signaling and suppressing the Pgr-Ihh axis.