(201) Endometrial Stromal PGR Signaling Affects Pregnancy and Glandular Hormone Responsiveness in the Uterus

Tancia Bradshaw Ph.D.¹; Sylvia Hewitt¹; Frank Orelana¹; Myeonjin Yi Ph.D¹; Barbara Nicol Ph.D.¹; Humphrey Yao Ph.D.¹; John Lydon Ph.D.²; Francesco DeMayo Ph.D¹

1. Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Science, Research Triangle Park, NC 27709


2.  Department of Molecular and Cell Biology, Baylor College of Medicine, Houston TX 77030

 

Abstract Text:

Progesterone, acting through its receptors, PGRA and PGRB, plays a major role in the establishment and maintenance of pregnancy by coordinating paracrine communication between the epithelium and stroma in the endometrium. Studies utilizing gene deletion of Pgr demonstrate that Pgr is required for embryo implantation, stromal proliferation and decidualization, and the inhibition of epithelial proliferation. Moreover, epithelial-specific deletion of Pgr phenocopies global knockout while overexpression impairs implantation. However, investigation of the phenotype of the genetic ablation of Pgr in the endometrial stroma have yet to be investigated.  Here we conducted endometrial stroma specific ablation of the Pgr to define the role of stroma Pgr in uterine function. Ablation of endometrial stroma Pgr was accomplished by crossing Pgr^f/f mice to Foxl2^Cre to generate Pgr^sd/sd mice. Immunohistochemistry confirmed the stroma-specific ablation of Pgr. Mice were then assayed for fertility, embryo implantation, the ability to undergo an artificially induced decidual response, hormonal induction of epithelial and stromal proliferation and the impact of the ablation on the uterine transcriptome at pregnancy day 3.5. Phenotypic analysis of the Pgr^sd/sd mice demonstrated female sterility with a failure of embryo implantation and uterine decidualization. Measurement of stromal and epithelial cell proliferation at PD 3.5 by Ki67 immunohistochemistry demonstrated the Pgr^sd/sd mice had failure of normal stroma proliferation. However, epithelial cell proliferation was inhibited as in the control mice. The Foxl2^Cre model also ablates genes in the pituitary and ovary. Ovulation was confirmed by the presence of embryos in the uterus. However, serum progesterone levels were significantly lower but above the pregnancy threshold suggesting a potential pituitary-luteal phenotype. To determine if the impact on stroma and epithelial proliferation was not due to an ovarian defect, proliferation was measured in response to Estrogen (E2) and progesterone (P4). This analysis demonstrated that E2 and P4 treatment of Pgr^sd/sd mice showed that ablation of stroma Pgr resulted in impairment of stroma proliferation but did not impact the inhibition of epithelial proliferation. RNA sequence analysis was used to identify the impact stroma ablation on the PD 3.5 transcriptome. This analysis demonstrated that ablation of stroma Pgr resulted in impairment of the LIF signaling pathway required for embryo implantation. Further analysis demonstrated that the expression of the uterine specific phenotype in female mice failing artificial induction of key glandular epithelial markers such as Prss29, Spink1/3 and Foxa2, which were significantly reduced despite having normal number of uterine glands. This analysis demonstrated that stroma PGR is critical in the regulation of normal glandular cell differentiation.  This work demonstrates the importance of endometrial stroma signaling in the regulation of uterine homeostasis. Here, we show that Pgr is required in the endometrial stroma for endometrial stroma proliferation, endometrial decidualization, and for the first time, endometrial glandular hormone responsiveness.