Uterine epithelial ERa regulates preimplantation uterine luminal epithelial (LE) and uterine mRNAs

Uterine epithelial estrogen receptor a (ERa) deficiency in epiERa-/- (Esr1f/-Wnt7aCre/+) mice leads to dysregulated environment of uterine lumen, which is lined by uterine luminal epithelium (LE). We hypothesized that LE transcriptomes held molecular keys to understanding ERa in regulating preimplantation uterine environment. Day 0.5 post-coitum (D0.5) and D3.5 LE (isolated via enzymatic digestion), digested uterus (DU), and flash-frozen uterus (U) from Esr1f/- (control) and epiERa-/- mice were processed for mRNA-seq. There were minimal differentially expressed genes (DEGs, TPM>1, FC>2, FDR<0.05) between DU and U. Between D0.5 and D3.5 Esr1f/- LE, the top upregulated and downregulated GOBP pathways were on cellular processes and on innate immune responses, respectively, with the later also seen in Esr1f/- U. Compared to Esr1f/- LE, the most upregulated and downregulated pathways in D0.5 epiERa-/- LE were on innate immune responses and on biosynthesis and metabolism, respectively; while those in D3.5 epiERa-/- LE were on cell division and on signaling and metabolic processes, respectively. Na+ transmembrane transport was among shared upregulated pathways in D0.5 and D3.5 epiERa-/- LE. Between Esr1f/- and epiERa-/-, most DEGs in U were also DEGs in LE, limited DEGs in U only with higher expression than in LE were related to immune responses, implicating potential paracrine effects of uterine epithelial ERa. Selected DEGs were verified by realtime PCR and immunohistochemistry. This mRNA-seq dataset provides molecular keys to understanding temporal (e.g., innate immunity) and constituent (e.g., uterine fluid movement) functions, and potential paracrine effects of uterine epithelial ERa in regulating the preimplantation uterine environment. Overall design: Virgin Esr1f/- and epiERa-/- female mice at ~2-6 months old were mated with wild type stud males (Esr1f/f or C57BL/6). They were checked every morning for the presence of a vaginal plug, an indication of mating activity during the previous night. The day of plug identification is defined as day 0.5 post-coitum (D0.5). The plugged females were separated from the stud males and randomly assigned into D0.5 and D3.5 time points, with same genotype littermates assigned into different timepoint groups. There were four groups of mice: D0.5 Esr1f/- group, D0.5 epiERa-/- group, D3.5 Esr1f/- group, and D3.5 epiERa-/- group. The mice were dissected at 11~12 h on both days. For each uterus, ¼ of one uterine horn was fixed in 10% formalin and ¼ of the other uterine horn was flash-frozen on dry-ice, designated as “U” (uterus), the remaining ¾ uterine horns connected by the cervix were slit open, immersed in 0.5% dispase (REF#17105-041, gibco, Waltham, MA, USA) in 1xHBSS buffer without calcium or magnesium (REF#1415-052, gibco, Waltham, MA, USA) for 30 min in a 6-cm culture dish, modified from our previous procedure (3-5). To determine the effects of enzymatic digestion process on uterine gene expression, we collected a small piece of digested uterine tissue prior to being processed for LE isolation. The small piece of digested uterine tissue was frozen on dry-ice and designated as “DU” (digested uterus). The LE layer in the remaining uterine tissue was gently scraped. The dislodged LE sheets were collected into a 1.5 ml microcentrifuge tube using a 200 µl pipette tip under a dissection microscope, centrifuged at 2000g for 3 minutes at 4C. Upon the removal of the buffer, 1 ml of TRIzol (REF# 15596018, Invitrogen, Carlsbad, CA, USA) was added to the LE pellet, which was gently vortexed to disperse the LE pellet, and kept at -20C. The U and DU tissues were carefully powdered in liquid N2 using a mortar and pestle, collected into a 1.5 ml microcentrifuge tube, in which 1 ml of TRIzol was added, and upon gentle vortexing to disperse the uterine powder, the tube was kept at -20C. At the conclusion of tissue collection, the LE, DU, and U samples in TRIzol were processed as one batch per tissue for total RNA isolation.