(177) In vivo Interplay of Ovarian Hormones and Small GTPase RhoA in Mouse Uterus

Jackson Kyle Sundgren^1,, Taylor Elijah Martin^1,2,, Yuehuan Li^{1 ,}, Jonathan Matthew Hancock^1,2, , and Xiaoqin Ye^1,2

< !^{1.      Department of Physiology and Pharmacology, University of Georgia, Athens, GA}

^{22. Interdisciplinary Toxicology Program, University of Georgia, Athens, GA}

Abstract Text:

Embryo implantation is an essential and rate-limiting step for mammalian pregnancy. It requires timely mutual communications between a competent embryo and a transiently receptive uterus, which is under the control of ovarian hormones progesterone (P4) and estrogen (E2). The first physical embryo-maternal interaction is established between trophoblasts and uterine luminal epithelial (LE) cells during embryo implantation initiation. How the LE senses the presence of an embryo in preparation for implantation remains largely unknown. Our previous study revealed small GTPase RhoA (Ras homolog family member A) as one of the most highly expressed genes in the day 3.5 post-coitum (D3.5) and D4.5 mouse LE (implantation initiates ~D4.0 in mice). RhoA is a prominent member of the Rho family of GTPases, which can serve as mechano-sensors. It regulates fundamental cellular functions, such as cytoskeletal organization and cell-extracellular matrix (ECM) interaction. Minimal defined in vivo functions of RhoA in the uterus have been investigated. RhoA is intensely detected in the apical membrane of D3.5 LE, the initial contact of an implanting embryo. Since global deletion of RhoA in mice is embryonic lethal and our attempts to generate uterine epithelial conditional knockout mouse models failed (RhoA^f/fWnt7a^Cre/+ mice died postnatally; and both Ltf and RhoA are in chromosome 9 which  prevents the generation of RhoA^f/fLtf^Cre/+ mice), we generated RhoA conditional knockout RhoA^f/fPgr^Cre/+ mice (RhoA^d/d), in which RhoA is deleted in progesterone receptor (PR)-expressing cells, the majority of cells in the female reproductive tract. Despite normal ovulation, RhoA^d/d mice have P4 deficiency caused by deletion of RhoA in luteal cells. Additionally, these mice are infertile due to failed embryo implantation. To circumvent this confounding factor, we treated preimplantation RhoA^d/d mice with P4, which partially rescued embryo implantation. These observations suggest potential local functions of RhoA in the uterus, which are being investigated in ovariectomized RhoA^f/fPgr^+/+ (control) and RhoA^d/d mice treated with vehicle, P4, E2, or E2 + P4 for 24 hours. Pilot PAS staining (detecting mucins, etc.) data show basal staining and responses to these treatments in both control and RhoA^d/d uteri. Pilot Masson’s Trichrome staining, which detects collagen (the dominant protein in ECM), etc., reveals comparable staining in vehicle-treated control and RhoA^d/d uteri, but upon P4 treatment, there is enhanced staining in the RhoA^d/d uteri (largely in intracellular space and extracellular matrices) compared to that in the control uteri. P4 signaling regulates collagen levels. These preliminary data suggest potential functions of uterine RhoA in mediating P4-regulated uterine functions. Since the uterine effects of P4 are mainly mediated via PR, a transcription factor, we are collecting uterine tissues from vehicle, P4, E2, or E2 + P4-treated ovariectomized RhoA^f/fPgr^+/+ and RhoA^d/d mice for mRNA-seq, which will provide a comprehensive picture of RhoA in regulating uterine transcriptome. Selected differentially expressed genes in the RhoA^d/d uterus will be further analyzed to elucidate the molecular mechanisms of RhoA in regulating uterine functions.